Prothrombotic status in Myeloproliferative neoplasms : the role of JAK2V617F allele burden and platelets/leukocytes activation

Dissertação de mestrado em Biologia Celular e Molecular apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de CoimbraAs neoplasias mieloproliferativas (MPN) são doenças da “stem-cell” hematopoiética e que estão associadas à ocorrência de eventos trombohemorragicos. Diferentes estudos sugerem que doentes com Policitémia Vera (PV) e Trombocitémia Essencial (ET) apresentam um estado protrombótico e que este poderá estar relacionado não só com a activação constitutiva da via JAK/STAT mas também com a carga alélica da mutação JAK2V617F. Objectivo: Investigar a presença de marcadores de activação hemostática e a relação destes com a carga alélica JAK2V617F e trombose. Métodos: Foram estudados 28 PV, 47 ET e 48 controlos saudáveis. Os doentes estão clinicamente estáveis e sob tratamento com Hidroxiureia; tempo de seguimento de 78 meses nas PV e de 84 meses nas ET. Sete doentes com PV e 16 com ET apresentaram história de trombose ao diagnóstico. Após consentimento informado, os doentes suspenderam a aspirina nos 10 dias prévios ao estudo. Por citometria de fluxo (FACSCalibur, BD) avaliou-se: expressão de P-selectina (CD62P) e granulofisina (CD63) plaquetar, basal e após estímulo com agonistas; capacidade de captação e libertação de mepacrina nas plaquetas; agregados plaqueta-leucócito; expressão de CD11b nos leucócitos e de factor tecidular (TF) nos monócitos, basal e após estímulo com LPS. A pesquisa da mutação JAK2V617F foi efectuada por PCR alelo específica e a quantificação por PCR em Tempo Real (JAK2 MutaQuant, Ipsogen). O screening de mutações no exão 10 do gene MPL, foi efectuado por SSCP e as mutações identificadas por sequenciação (ABI 310 Genetic Analyzer, AB). Resultados: A mutação JAK2V617F foi encontrada em 28 PV (100%) e 28 ET (60%); 2 doentes com ET, apresentam mutações no exão 10 do gene MPL: W515L e R524C. Os doentes apresentam um aumento significativo de expressão basal de CD62P e de CD63 e de resposta ao ácido araquidónico; em todos os doentes a resposta ao TRAP6 está significativamente diminuída; 77% das PV e 50% das ET apresentam um fenótipode “storage pool disease”. Todos os doentes apresentam um aumento significativo de expressão basal de CD11b nos leucócitos e de TF nos monócitos. Os agregados plaqueta-leucócito estão significativamente aumentados em todos os doentes, sendo que os agregados plaqueta-neutrófilo (PMN) estão significativamente aumentados nas ET vs PV. Os doentes com carga alélica JAK2V617F>50% apresentam um aumento significativo na expressão de CD11b nos leucócitos e de agregados plaqueta-PMN, em comparação com os doentes com carga alélica <50%; as PV com carga alélica >50% apresentam um aumento estatisticamente significativo de TF nos monócitos. Nas ET, foi encontrada uma correlação estatisticamente significativa entre trombose e a presença de mutações JAK2V617F ou MPL, e entre trombose e carga alélica>50%. Esta associação não foi encontrada nas PV. A relação entre carga alélica e alterações plaquetares é difícil de estabelecer, uma vez que não foram encontradas alterações estatisticamente significativas. Discussão: Os dados apresentados mostram, com significado estatístico, que os doentes com PV e ET apresentam plaquetas e leucócitos activados e um aumento de agregados plaqueta-leucócito em circulação. Os doentes com carga alélica>50% apresentam marcadores de activação significativamente aumentados em comparação com os doentes com carga alélica <50%, consistente com a influência da carga alélica e perda de heterozigotia para a mutação JAK2V617F na activação celular. Nas ET a presença da mutação e a carga alélica >50% está significativamente associada a eventos trombóticos ao diagnóstico. Conclusão: Uma vez que as tromboses representam, nos doentes com MPN, uma das principais causas de co-morbilidade, foram investigados marcadores de activação da hemostase e a sua relação com a carga alélica JAK2V617F. Os resultados apresentados ilustram diferentes mecanismos que favorecem a trombose nas PV e ET, nomeadamente, activação basal das plaquetas, monócitos e PMN, aumento de FT nos monócitos e de aumento de agregados plaqueta-leucócito em circulação.Introduction: Myeloproliferative neoplasms (MPN) are stem cell-derived proliferative diseases associated with thrombohemorragic diathesis. Different studies suggested that Polycythemia Vera (PV) and Essential Thrombocythemia (ET) patients have a baseline protrombotic status, which could be related to constitutive JAK2/STAT signalization in correlation with JAK2V617F mutation allele burden. Objective: Investigate the baseline hemostatic activation markers and their correlation with the JAK2V617F allele burden and thrombosis. Methods: 28 PV and 47 ET patients and a control group of 48 healthy volunteers were studied. All the patients are under hydroxyurea treatment and remain clinically stable with follow up periods of 78 months for PV and 84 months for ET. Seven PV and 16 ET patients have a history of thrombosis at diagnosis. With patients’ written informed consent, aspirin was withdrawn for 10 days prior the studies. Using flow cytometry (FACSCalibur, BD), we evaluated: platelet P-selectin (CD62P) and granulophysin (CD63), at baseline and after stimulation with agonists; platelet uptake and release of mepacrine; platelet-leukocyte aggregates, leukocyte CD11b and monocyte Tissue factor (TF) at baseline and after stimulation with LPS. JAK2V617F allele was detected by Allele specific PCR and quantified by Allele specific Real Time PCR (JAK2 MutaQuant, Ipsogen). MPL exon 10 mutations were screened by SSCP and identified by direct sequencing (ABI 310 Genetic Analyzer, Applied Biosystems). Results: JAK2V617F mutation was found in 28 PV patients (100%) and in 28 ET patients (60%). In two other ET patients we found MPL gene exon 10 mutations: W515L and R524C. All patients have, at baseline, a significant increased expression of CD62P and CD63, and a significant increase response to arachidonic acid and diminished levels of CD62P and CD63 following TRAP6 activation. A phenotype of storage pool disease was found in 77% of PV and 50% of ET patients. At baseline all patients have activated leukocytes with a statistically significant increase in CD11bexpression and monocyte-TF. The latter was significantly elevated in PV vs ET patients. Circulating platelet-leukocytes aggregates were significant higher in all patients; in the ET, platelet-PMN aggregates were significantly increased vs PV patients. Patients with JAK2V617F>50%, have a statistically significant increase in leukocytes CD11b expression and in platelet PMN-aggregates, in comparison with those with JAK2V617F50% have a statistically significant increase in monocytes-TF. In ET patients we found a statistically significant correlation between thrombosis and JAK2 or MPL mutations, furthermore, ET patients with JAK2V617F allele burden >50% have statistically higher incidence of thrombosis. No such correlation in PV patients. Regarding to platelets activation, it’s not evident such effect since no significantly differences were found. Discussion: These data shown, with statistically significance that PV and ET patients have circulating activated platelets and leukocytes, and increased number of plateletleukocyte aggregates. Consistent with the influence of allele burden and acquisition of loss of heterozygosity for the JAK2V617F mutation in leukocyte activation, all patients with allele burden >50% presented significantly increased activation markers comparing to patients with allele burden >50%. In ET patients the presence of JAK2V617F mutation and allele burden>50% was significantly associated with a previous history of thrombosis. Conclusion: As thrombosis is one of the main co-morbilities in MPN patients, we investigated the baseline hemostatic activation markers and their correlation with the JAK2V617F allele burden. These data clearly illustrate several mechanisms favoring thrombosis in PV and ET patients, namely, the baseline activation of platelets, monocytes and PMNs leukocytes, increased monocyte-TF and platelet-leukocytes aggregates

Negative MR4·0 chronic myeloid leukaemia and its possible implications for treatment-free remission

© 2019 British Society for Haematology and John Wiley & Sons Ltd.ABL1 tyrosine kinase inhibitors (TKI) have dramatically improved the outcome for chronic myeloid leukaemia (CML) patients, resulting in a life expectancy that approaches that of the general population. Nevertheless, lifelong TKI therapy may have consequences, including chronic adverse events that can substantially impact patients’ quality of life, adherence to therapy and treatment success. Recently, several clinical discontinuation trials have demonstrated that 40–60% of chronic phase CML patients (CP-CML) who have achieved a stable deep molecular response (DMR) can stop therapy without relapsing (Breccia & Foà, 2018). Laboratory recommendations for scoring DMR were previously defined as MR4·0 [either detectable disease ⩽0·01% BCR-ABLIS (MR4·0 positive) or undetectable disease in cDNA with 10 000–31 999 ABL1 transcripts or 24 000–76 999 GUSB transcripts (MR4·0 negative)], MR4·5 [either detectable disease ⩽0·0032% BCR-ABLIS (MR4·5 positive) or undetectable disease in cDNA with 32 000–99 999 ABL1 transcripts or 77 000–239 999 GUSB transcripts (MR4·5 negative)], and MR5·0 [either detectable disease ⩽0·001% BCR-ABLIS (MR5·0 positive) or undetectable disease in cDNA with ⩾100 000 ABL1 transcripts or ⩾240 000 GUSB transcripts (MR5·0 negative)] (Cross et al, 2015).info:eu-repo/semantics/publishedVersio

Genetic variants of ABC and SLC transporter genes and chronic myeloid leukaemia: impact on susceptibility and prognosis

Solute carrier (SLC) and ATP-binding cassette (ABC) transporters comprise a variety of proteins expressed on cell membranes responsible for intrusion or extrusion of substrates, respectively, including nutrients, xenobiotics, and chemotherapeutic agents. These transporters mediate the cellular disposition of tyrosine kinase inhibitors (TKIs), and their genetic variants could affect its function, potentially predisposing patients to chronic myeloid leukaemia (CML) and modulating treatment response. We explored the impact of genetic variability (single nucleotide variants—SNVs) of drug transporter genes (ABCB1, ABCG2, SLC22A1, and SLC22A5) on CML susceptibility, drug response, and BCR-ABL1 mutation status. We genotyped 10 SNVs by tetra-primers-AMRS-PCR in 198 CML patients and 404 controls, and assessed their role in CML susceptibility and prognosis. We identified five SNVs associated with CML predisposition, with some variants increasing disease risk, including TT genotype ABCB1 (rs1045642), and others showing a protective effect (GG genotype SLC22A5 rs274558). We also observed different haplotypes and genotypic profiles associated with CML predisposition. Relating to drug response impact, we found that CML patients with the CC genotype (rs2231142 ABCG2) had an increased risk of TKI resistance (six-fold). Additionally, CML patients carrying the CG genotype (rs683369 SLC22A1) presented a 4.54-fold higher risk of BCR-ABL1 mutations. Our results suggest that drug transporters’ SNVs might be involved in CML susceptibility and TKI response, and predict the risk of BCR-ABL1 mutations, highlighting the impact that SNVs could have in therapeutic selection.info:eu-repo/semantics/publishedVersio

CD20+ T cells in monoclonal B cell lymphocytosis and chronic lymphocytic leukemia: frequency, phenotype and association with disease progression

IntroductionIn monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), the expansion of malignant B cells disrupts the normal homeostasis and interactions between B cells and T cells, leading to immune dysregulation. CD20+ T cells are a subpopulation of T cells that appear to be involved in autoimmune diseases and cancer.MethodsHere, we quantified and phenotypically characterized CD20+ T cells from MBL subjects and CLL patients using flow cytometry and correlated our findings with the B-cell receptor mutational status and other features of the disease. Results and discussionCD20+ T cells were more represented within the CD8+ T cell compartment and they showed a predominant memory Tc1 phenotype. CD20+ T cells were less represented in MBL and CLL patients vs healthy controls, particularly among those with unmutated IGVH gene. The expansion of malignant B cells was accompanied by phenotypic and functional changes in CD20+ T cells, including an increase in follicular helper CD4+ CD20+ T cells and CD20+ Tc1 cells, in addition to the expansion of the TCR Vβ 5.1 in CD4+ CD20+ T cells in CLL

Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next-generation sequencing

BACKGROUND: The 2 main forms of thrombotic microangiopathy (TMA) are thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). Deficiency of ADAMTS13 and dysregulation of the complement pathway result in TTP and aHUS, respectively; however, overlap of their clinical characteristics makes differential diagnosis challenging. OBJECTIVES AND METHODS: We aimed to develop a TMA diagnosis workflow based on ADAMTS13 activity and screening of ADAMTS13 and complement genes using a custom next-generation sequencing (NGS) gene panel. PATIENTS: For this, from a cohort of 154 Portuguese patients with acute TMA, the genotype-phenotype correlations were analyzed in 7 hereditary TTP (ADAMTS13 activity <10%, no inhibitor), 36 acquired TTP (ADAMTS13 activity <10%, presence of an inhibitor), and in 34 presumable aHUS. RESULTS: In total, 37 different rare variants, 8 of which novel (in ADAMTS13,CFH, and CD46), were identified across 7 genes. Thirteen TTP patients were homozygous (n=6), compound heterozygous (n=2), and heterozygous (n=5) for 11 ADAMTS13 variants (6 pathogenic mutations). Among the 34 aHUS patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause aHUS only when combined with other variants and/or CFH and CD46 risk haplotypes or CFHR1-3 deletion. CONCLUSIONS: Our study provides evidence of the usefulness of the NGS panel as an excellent technology that enables more rapid diagnosis of TMA, and is a valuable asset in clinical practice to discriminate between TTP and aHUS.info:eu-repo/semantics/publishedVersio