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2.
Br J Haematol ; 196(3): 676-680, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34562020

RESUMO

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.


Assuntos
Eritropoetina/sangue , Janus Quinase 2/genética , Mutação , Policitemia Vera/sangue , Policitemia Vera/genética , Alelos , Substituição de Aminoácidos , Tomada de Decisão Clínica , Gerenciamento Clínico , Índices de Eritrócitos , Volume de Eritrócitos , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Volume Plasmático , Policitemia Vera/diagnóstico , Policitemia Vera/epidemiologia , Sensibilidade e Especificidade
3.
Haematologica ; 105(6): 1539-1551, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31488557

RESUMO

Myelodysplastic syndromes and acute myeloid leukemia with TP53 mutations are characterized by frequent relapses, poor or short responses, and poor survival with the currently available therapies including chemotherapy and 5-azacitidine (AZA). PRIMA-1Met(APR-246,APR) is a methylated derivative of PRIMA-1, which induces apoptosis in human tumor cells through restoration of the transcriptional transactivation function of mutant p53. Here we show that low doses of APR on its own or in combination with AZA reactivate the p53 pathway and induce an apoptosis program. Functionally, we demonstrate that APR exerts these activities on its own and that it synergizes with AZA in TP53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) cell lines and in TP53-mutated primary cells from MDS/AML patients. Low doses of APR on its own or in combination with AZA also show significant efficacy in vivo Lastly, using transcriptomic analysis, we found that the APR + AZA synergy was mediated by downregulation of the FLT3 pathway in drug-treated cells. Activation of the FLT3 pathway by FLT3 ligand reversed the inhibition of cell proliferation by APR + AZA. These data suggest that TP53-mutated MDS/AML may be better targeted by the addition of APR-246 to conventional treatments.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Compostos Aza , Azacitidina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Quinuclidinas , Proteína Supressora de Tumor p53/genética
4.
Ann Hematol ; 98(1): 111-118, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30259120

RESUMO

The JAK2V617F mutation is part of the major criteria for diagnosis of myeloproliferative neoplasms (MPN). Allele-specific quantitative PCR (qPCR) is the most prevalent method used in laboratories but with the advent of next-generation sequencing (NGS) techniques, we felt necessary to evaluate this approach for JAK2 mutations testing. Among DNA samples from 427 patients analyzed by qPCR and NGS, we found an excellent concordance between both methods when allelic burden was superior to 2% (the detection limit of our NGS assay). Only one sample among 298 was found negative by NGS while allelic burden by qPCR was 3%. Because NGS detection limit is higher, sensitivity was lower as exemplified by 21 samples found negative whereas qPCR measured allelic burdens between 0.1 and 1%. Importantly, quantitative data of samples found positive by both techniques were highly correlated (R2 = 0.9477). We also evaluated 40 samples tested for JAK2 exon 12 mutations by HRM. The concordance with NGS was of 100%. Using NGS, the full coding region of JAK2 was analyzed leading to identification of several variants outside of exon 12 and 14 which were previously described or not. Interestingly, we found one somatic mutation (c.1034A>T p.H345L) which induced constitutive activation of the JAK/STAT pathway leading to an increased proliferation of BaF/3 cells with low-dose EPO. This study showed that NGS is a robust method highly correlated to qPCR, although less sensitive, but providing the opportunity to identify other JAK2 variants with potential impact on disease initiation or evolution.


Assuntos
Éxons , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Substituição de Aminoácidos , Linhagem Celular Tumoral , Análise Mutacional de DNA/métodos , Feminino , Humanos , Janus Quinase 2/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real/métodos
6.
Blood ; 126(24): 2585-91, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26486786

RESUMO

Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.


Assuntos
Calreticulina/genética , Interferon-alfa/uso terapêutico , Mutação , Polietilenoglicóis/uso terapêutico , Trombocitemia Essencial/tratamento farmacológico , Adolescente , Adulto , Alelos , Aspirina/uso terapêutico , Evolução Clonal/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Dioxigenases , Feminino , Seguimentos , Genes p53 , Humanos , Hidroxiureia/uso terapêutico , Interferon-alfa/efeitos adversos , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Uso Off-Label , Polietilenoglicóis/efeitos adversos , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Indução de Remissão , Proteínas Repressoras/genética , Trombocitemia Essencial/sangue , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia , Adulto Jovem
8.
Blood Adv ; 8(12): 3330-3343, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38386979

RESUMO

ABSTRACT: Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for <1% of strokes resulting in brain parenchymal injuries. JAK2V617F mutation, the most frequent driving mutation of myeloproliferative neoplasms, has been reported to be associated with worse clinical outcomes in patients with CVST. We investigated whether hematopoietic JAK2V617F expression predisposes to specific pathophysiological processes and/or worse prognosis after CVST. Using an in vivo mouse model of CVST, we analyzed clinical, biological, and imaging outcomes in mice with hematopoietic-restricted Jak2V617F expression, compared with wild-type Jak2 mice. In parallel, we studied a human cohort of JAK2V617F-positive or -negative CVST. Early after CVST, mice with hematopoietic Jak2V617F expression had increased adhesion of platelets and neutrophils in cerebral veins located in the vicinity of CVST. On day 1, Jak2V617F mice had a worse outcome characterized by significantly more frequent and severe intracranial hemorrhages (ICHs) and higher mortality rates. Peripheral neutrophil activation was enhanced, as indicated by higher circulating platelet-neutrophil aggregates, upregulated CD11b expression, and higher myeloperoxydase plasma level. Concurrently, immunohistological and brain homogenate analysis showed higher neutrophil infiltration and increased blood-brain barrier disruption. Similarly, patients with JAK2V617F-positive CVST tended to present higher thrombotic burden and had significantly higher systemic immune-inflammation index, a systemic thromboinflammatory marker, than patients who were JAK2V617F-negative. In mice with CVST, our study corroborates that Jak2V617F mutation leads to a specific pattern including increased thrombotic burden, ICH, and mortality. The exacerbated thromboinflammatory response, observed both in mice and patients positive for JAK2V617F, could contribute to hemorrhagic complications.


Assuntos
Inflamação , Janus Quinase 2 , Mutação , Trombose dos Seios Intracranianos , Animais , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Trombose dos Seios Intracranianos/genética , Humanos , Prognóstico , Inflamação/genética , Modelos Animais de Doenças , Masculino , Feminino , Neutrófilos/metabolismo
9.
Leukemia ; 38(2): 326-339, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38148396

RESUMO

Current recommended risk scores to predict thrombotic events associated with myeloproliferative neoplasms (MPN) do not discriminate between arterial and venous thrombosis despite their different physiopathology. To define novel stratification systems, we delineated a comprehensive landscape of MPN associated thrombosis across a large long-term follow-up MPN cohort. Prior arterial thrombosis, age >60 years, cardiovascular risk factors and presence of TET2 or DNMT3A mutations were independently associated with arterial thrombosis in multivariable analysis. ARTS, an ARterial Thrombosis Score, based on these four factors, defined low- (0.37% patients-year) and high-risk (1.19% patients-year) patients. ARTS performance was superior to the two-tiered conventional risk stratification in our training cohort, across all MPN subtypes, as well as in two external validation cohorts. Prior venous thrombosis and presence of a JAK2V617F mutation with a variant allelic frequency ≥50% were independently associated with venous thrombosis. The discrimination potential of VETS, a VEnous Thrombosis Score based on these two factors, was poor, similar to the two-tiered conventional risk stratification. Our study pinpoints arterial and venous thrombosis clinico-molecular differences and proposes an arterial risk score for more accurate patients' stratification. Further improvement of venous risk scores, accounting for additional factors and considering venous thrombosis as a heterogeneous entity is warranted.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Trombose , Trombose Venosa , Humanos , Pessoa de Meia-Idade , Neoplasias/complicações , Trombose Venosa/genética , Trombose/genética , Trombose/complicações , Mutação , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/genética , Fatores de Risco , Janus Quinase 2/genética , Medição de Risco
11.
Blood Adv ; 6(9): 2813-2823, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35030630

RESUMO

The mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown, but TP53 mutations acquisition is considered a key event in this process. p53 is a main tumor suppressor, but mutations in this protein per se do not confer a proliferative advantage to the cells, and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However, the impact of these drugs on TP53-mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context, we used a DNA single-cell approach to determine the clonal architecture of TP53-mutated cells. We observed that TP53 mutations are late events in MPN, mainly occurring in the driver clone, whereas clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single-cell level, the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.


Assuntos
Antineoplásicos , Transtornos Mieloproliferativos , Células Clonais/metabolismo , Humanos , Mutação , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Análise de Célula Única , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
13.
Indian J Med Res ; 134: 532-7, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22089617

RESUMO

The first coherent pathophysiological scheme for sickle cell disease (SCD) emerged in the sixties-seventies based on an extremely detailed description of the molecular mechanism by which HbS in its deoxy-form polymerises and forms long fibres within the red blood cell that deform it and make it fragile. This scheme explains the haemolytic anaemia, and the mechanistic aspects of the vaso-occlusive crises (VOCs), but, even though it constitutes the basic mechanism of the disease, it does not account for the processes that actually trigger VOCs. This paper reviews recent data which imply: red blood cell dehydration, its abnormal adhesion properties to the endothelium, the participation of inflammatory phenomenon and of a global activation of all the cells present in the vessel, and finally, abnormalities of the vascular tone and of nitric oxide metabolism. These data altogether have shed a new light on the pathophysiology of the first molecular disease i.e. sickle cell disease.


Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/metabolismo , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemoglobina Falciforme/metabolismo , Adesão Celular , Endotélio Vascular/metabolismo , Hemoglobina Falciforme/genética , Hemólise , Humanos , Canais Iônicos/metabolismo , Óxido Nítrico/metabolismo
14.
J Exp Med ; 218(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33075130

RESUMO

Interferon α (IFNα) is used to treat JAK2V617F-driven myeloproliferative neoplasms (MPNs) but rarely clears the disease. We investigated the IFNα mechanism of action focusing on PML, an interferon target and key senescence gene whose targeting by arsenic trioxide (ATO) drives eradication of acute promyelocytic leukemia. ATO sharply potentiated IFNα-induced growth suppression of JAK2V617F patient or mouse hematopoietic progenitors, which required PML and was associated with features of senescence. In a mouse MPN model, combining ATO with IFNα enhanced and accelerated responses, eradicating MPN in most mice by targeting disease-initiating cells. These results predict potent clinical efficacy of the IFNα+ATO combination in patients and identify PML as a major effector of therapy, even in malignancies with an intact PML gene.


Assuntos
Trióxido de Arsênio/farmacologia , Interferon-alfa/farmacologia , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Proteína da Leucemia Promielocítica/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Mieloproliferativos/metabolismo
15.
Blood Adv ; 5(5): 1442-1451, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33666653

RESUMO

We aimed to study the prognostic impact of the mutational landscape in primary and secondary myelofibrosis. The study included 479 patients with myelofibrosis recruited from 24 French Intergroup of Myeloproliferative Neoplasms (FIM) centers. The molecular landscape was studied by high-throughput sequencing of 77 genes. A Bayesian network allowed the identification of genomic groups whose prognostic impact was studied in a multistate model considering transitions from the 3 conditions: myelofibrosis, acute leukemia, and death. Results were validated using an independent, previously published cohort (n = 276). Four genomic groups were identified: patients with TP53 mutation; patients with ≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1, IDH2, NRAS, or KRAS (high-risk group); patients with ASXL1-only mutation (ie, no associated mutation in TP53 or high-risk genes); and other patients. A multistate model found that both TP53 and high-risk groups were associated with leukemic transformation (hazard ratios [HRs] [95% confidence interval], 8.68 [3.32-22.73] and 3.24 [1.58-6.64], respectively) and death from myelofibrosis (HRs, 3.03 [1.66-5.56] and 1.77 [1.18-2.67], respectively). ASXL1-only mutations had no prognostic value that was confirmed in the validation cohort. However, ASXL1 mutations conferred a worse prognosis when associated with a mutation in TP53 or high-risk genes. This study provides a new definition of adverse mutations in myelofibrosis with the addition of TP53, CBL, NRAS, KRAS, and U2AF1 to previously described genes. Furthermore, our results argue that ASXL1 mutations alone cannot be considered detrimental.


Assuntos
Mielofibrose Primária , Teorema de Bayes , Genômica , Humanos , Mutação , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/genética , Prognóstico , Proteínas Repressoras/genética
16.
Pharmacogenet Genomics ; 20(4): 257-68, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20216336

RESUMO

BACKGROUND AND OBJECTIVE: The clinical hallmarks of sickle cell disease (SCD) are vaso-occlusive crises (VOC) triggered by red blood cells (RBC) stiffening and abnormal adhesion to vascular endothelial cells (VEC) in the context of chronic inflammation, cell activation, and vascular tone abnormalities. Hydroxycarbamide (HC) is the only drug with a proven efficacy in decreasing VOC frequency. HC decreases RBC stiffening, modulates adhesion protein expression by RBC and VEC, and reduces endothelin-1 production by VEC. Our objective was to test whether HC could also affect inflammation through its action on VEC. METHODS: We used microarrays to study the effect of HC on the transcriptome of transformed human bone marrow endothelial cell, a cell line derived from bone marrow microcirculation (the predilection site of VOC), in basal and proinflammatory conditions. Microarray results were confirmed by real-time quantitative PCR and protein analysis on transformed human bone marrow endothelial cell (TrHBMEC) and on two other VEC types in the primary culture: human pulmonary microcirculation endothelial cell (HPMEC) and human umbilical vein endothelial cell (HUVEC a classical model for the macrocirculation). RESULTS: HC had a significant effect on the expression of genes of the 'inflammation pathway'. Strikingly, it stimulates the expression of proinflammatory genes such as IL1A, IL1B, IL6, IL8, CCL2, CCL5, CCL20, and CCL8 in all the tested VEC types. CONCLUSION: Our study confirms that VECs are significant targets of HC in the context of SCD and identifies its earlier unsuspected action on another major component of SCD pathophysiology, that is, the 'inflammation pathway'.


Assuntos
Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Antidrepanocíticos/farmacologia , Citocinas/biossíntese , Hidroxiureia/farmacologia , Mediadores da Inflamação/metabolismo , Anemia Falciforme/genética , Linhagem Celular , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Citocinas/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Interleucinas/biossíntese , Interleucinas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Farmacogenética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Int J Lab Hematol ; 42(6): 827-832, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32614523

RESUMO

INTRODUCTION: The pathogenesis of myeloproliferative neoplasms (MPNs) is closely related to the acquisition of specific molecular alterations in JAK2, MPL, or CALR genes, the presence of which represent major diagnostic criteria in the WHO classification. The CALR exon 9 insertions and deletions are very heterogeneous, and their detection mainly relies on polymerase chain reaction (PCR) fragment length analysis. METHODS: We report on the rare nonclassical profiles that we observed among the 1382 patients analyzed by PCR fragment length analysis. In difficult cases, we tested germline DNA and performed NGS analysis. RESULTS: We faced some troubling results because of the presence of several unexpected peaks. Our investigations showed that they resulted from a mix of isolated or double somatic mutations combined with germline alterations which could be misleading for a correct diagnosis. The precise interpretation of such difficult cases is mandatory as a misinterpretation could lead to the prescription of cytoreductive drugs to nondiseased persons or to an absence of treatment in true MPN patients. CONCLUSION: Our observations showed that every mutation should be verified by direct Sanger sequencing, and we show that sometimes it may be necessary to study germline DNA and to complement with NGS analysis to precisely interpret the molecular alterations.


Assuntos
Calreticulina/genética , Éxons , Neoplasias Hematológicas/genética , Mutação , Transtornos Mieloproliferativos/genética , Proteínas de Neoplasias/genética , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
18.
Blood Adv ; 4(15): 3708-3715, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32777065

RESUMO

Myeloproliferative neoplasms (MPNs) are the most frequent underlying causes of splanchnic vein thromboses (SVTs). MPN patients with SVTs (MPN-SVT) often have a unique presentation including younger age, female predominance, and low Janus kinase 2 (JAK2) mutation allele burden. This study aimed at identifying risk factors for adverse hematologic outcomes in MPN-SVT patients. We performed a retrospective study of a fully characterized cohort of MPN-SVT patients. The primary outcome was the incidence of evolution to myelofibrosis, acute leukemia, or death. Eighty patients were included in the testing cohort. Median follow-up was 11 years. Most of the patients were women with a mean age of 42 years and a diagnosis of polycythemia vera. The primary outcome was met in 13% of the patients and was associated with a JAK2V617F allele burden ≥50% (odds ratio [OR], 14.7) and presence of additional mutations in genes affecting chromatin/spliceosome (OR, 9). We identified high-risk patients (29% of the cohort) as those harboring at least 1 molecular risk factor: JAK2-mutant allele burden ≥50%, presence of chromatin/spliceosome/TP53 mutation. High-risk patients had worse event-free survival (81% vs 100%; P = .001) and overall survival at 10 years (89% vs 100%; P = .01) than low-risk patients. These results were confirmed in an independent validation cohort of 30 MPN-SVT patients. In conclusion, molecular profiling identified MPN-SVT patients with dismal outcome. In this high-risk population, a disease-modifying therapy should be taken into consideration to minimize the probability of transformation.


Assuntos
Transtornos Mieloproliferativos , Policitemia Vera , Mielofibrose Primária , Trombose Venosa , Adulto , Feminino , Humanos , Masculino , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Estudos Retrospectivos
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