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.

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.

Thrombocythaemia is defined as a circulating platelet count above 450x10$^9$/L in humans. The major cause of thrombocythaemia is reactive $(secondary)$ thrombocythaemia which occurs secondary to many conditions such as infection, cancer and inflammation. However, acquired clonal mutations in mainly Janus Kinas 2 $(JAK2)$, CALR and MPL cause essential thrombocythaemia $(ET)$. ET is a rare disease that leads to an increased risk of cardiovascular thrombotic events. Current treatment of ET uses combination of low dose aspirin to decrease platelet function and cytoreductive agents to decrease thrombopoiesis. The most commonly used cytoreductive agents are hydroxyurea, anagrelide and interferon-$alpha$ and all have unwanted side effects. Cytokine receptor-like factor 3 $(CRLF3)$ is a 2.4kb gene that is ubiquitously expressed throughout the haematopoietic system. Very little is known about the function of CRLF3, with only one peer reviewed journal article in the literature which shows that CRLF3 may negatively regulate the cell cycle at the G0/G1 phase. However, nothing is known about the role of CRLF3 in platelet biology. Using a Crlf3 knockout mouse $(Crlf3-/-)$ developed by the Wellcome Trust Sanger Institute we show CRLF3’s role in platelet biogenesis and how it could be used as a novel therapeutic target to treat ET. Crlf3-/- mice have an isolated and sustained 25-40$\%$ decrease in platelet count compared to wildtype $(WT)$ controls. Platelet function is unaffected as demonstrated in a range in a range of in vitro assays. The thrombocytopenia is a consequence of abnormalities in hematopoietic cells, as shown by bone marrow transplantations. Megakaryopoiesis is upregulated in Crlf3-/- mice and proplatelet morphology is unaffected, suggesting the thrombocytopenia is due to increased platelet clearance. Indeed, splenectomised Crlf3-/- mice show normalised platelet counts within 7 days, showing rapid splenic removal of platelets is responsible for the thrombocytopenia. Abnormal large platelet structures that resemble proplatelets shafts $(preplatelets)$ are abnormally present in the circulation of elderly Crlf3/- mice. Immunohistochemistry showed increased and aberrant tubulin expression in Crlf3-/- platelets compared to WT controls, especially in the preplatelet forms. Cold induced depolymerisation of microtubules was decreased in Crlf3-/- platelets, suggestive of increased tubulin stability, however, the ratio of detyrosinated to tyrosinated tubulin was not altered. We then crossbred Crlf3-/- mice with JAK2 V617F ET mice, to determine the effect of Crlf3 ablation of thrombocythaemia. Crossbred mice showed restoration of platelet counts to WT values without grossly affecting platelet function or other blood lineages, providing the rational for CRLF3 as a novel therapeutic target for treatment of ET. Finally, we aimed to resolve the crustal structure of CRLF3 and discover its interactome. To this end, we were able to resolve the crystal structure of a C-terminal portion of the full length protein containing the predicted fibronectin type III domain. To shed light on the interactome of CRLF3, endogenous CRLF3 was tagged with a tandem affinity purification $(TAP)$ tag using CRISPR/Cas9 technology in induced pluripotent stem cells $(iPSCs)$. We have been able to produce megakaryocytes from these TAP-tagged iPSCs by forward programming. However, as yet we have not been able to generate enough MKs to have adequate material to perform immunoprecipitation assays. Therefore, the interactome of CRLF3 in MKs remains unknown. In conclusion, we identified a mechanism by which Crlf3 controls platelet biogenesis. Slowed maturation of Crlf3-/- preplatelets in the peripheral circulation potentially due to increased structural stability leads to rapid removal of these immature forms by the spleen and therefore a decrease in platelet count. The isolated effect on platelet numbers and normalisation of platelet count in ET mice deficient of Crlf3 provides the rational for further study on CRLF3 drug targeting as a novel therapeutic strategy for ET.

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.

Les syndromes myéloprolifératifs (SMP) non leucémie myéloïde chronique (LMC) sont des hémopathies myéloïdes chroniques affectant la cellule souche hématopoïétique, pouvant évoluer en leucémie aigüe myéloïde (LAM). Les SMP non LMC incluent la polyglobulie de Vaquez (PV), la thrombocytémie essentielle (TE) et la myélofibrose (MF). La mutation JAK2V617F est retrouvée dans 97% des cas de PV et dans 50% des cas de TE et MF ; elle n'est pas indispensable à la physiopathologie des SMP car JAK2 n'est pas muté à 100%. Afin de progresser dans la compréhension de la physiopathologie des SMP et afin d'identifier de nouveaux marqueurs moléculaires pour le diagnostic, le suivi et le pronostic; nous avons étudié des échantillons de PV, TE, MF ainsi que des LAM post-SMP. Nous avons utilisé des approches moléculaires complémentaires: séquençage, hybridation génomique comparative (CGH-array) et profils d'expression génique. Nous avons identifié des mutations de gènes régulateurs de l'épigénétique (ASXL1, TET2, DNMT3A, SUZ12) et des gènes de la machinerie de l'épissage de l'ARN (SF3B1, SRSF2). Nous avons également identifié que les co-mutations des gènes JAK2 et ASXL1 étaient associées à un mauvais pronostic. Au sein du sous-type MF, nous avons identifié par CGH-array des aberrations du nombre de copies des gènes. Celles-ci contiennent plusieurs gènes candidats susceptibles de participer à la physiopathologie des MF et à l'évolution en LAM (délétion 20q, NF1, ETV6). Nos travaux sur la caractérisation moléculaire des SMP contribuent à l'évolution vers une classification moléculaire avec l'objectif d'une médecine de précision où chaque SMP sera traité en fonction de ses altérations. / Myeloproliferative neoplasms (MPN) are chronic and clonal stem cell myeloid disorders, which can evolve to acute myeloid leukemia (AML). MPN non-chronic myeloid leukemia (CML) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) (primary or secondary to PV/ET). JAK2V617F mutation is found in 97% of PV and in around half of patients with ET or MF. Nevertheless, this mutation is not essential for MPN physiopathology, because in half of ET/MF cases, JAK2 is not mutated. To progress in the knowledge of MPN physiopathology and in particular of MF; and to find new molecular markers for MPN diagnosis, disease course, and prognosis, we studied several samples of PV, ET, MF and post-AML. We used gene sequencing, array-Comparative Genomic Hybridization (aCGH) and gene expression analyses. We identified several mutations in genes implicated in Epigenetic regulation (ASXL1, TET2, DNMT3A, SUZ12) and in genes implicated in the RNA splicing machinery (SF3B1, SRSF2). We also found that JAK2 and ASXL1 co-mutation is associated with a poor prognosis. In MF, we found by aCGH several copy number aberrations that involve potential leukemogenic genes. Our gene expression data support the hypothesis that PV, ET and MF are a continuum of the same pathology. Our results on molecular characterization help establish a new molecular classification of MPNs with the objective personalized treatment where each MPN will be treated depending on the alterations present in the myeloid cell genome.