Impacto clínico de la cuantificación de la carga mutacional de JAK2V617F en pacientes con neoplasias mieloproliferativas crónicas Ph negativas clásicas / Clinical significance of the quantification of JAK2V617F allele burden in classical Ph-negative myeloproliferative neoplasms

Fundamento y objetivo: La identificación de la mutación V617F del gen JAK2 en pacientes diagnosticados de neoplasias mieloproliferativas crónicas (NMPC) es fundamental en el cribado diagnóstico. Nuestro objetivo fue investigar la asociación entre la cuantificación de la mutación V617F (carga alélica JAK2V617F) y el fenotipo clínico al diagnóstico, y definir el papel de la carga alélica en la predicción de complicaciones. Pacientes y métodos: En el Servicio de Hematología del Hospital Universitario La Paz realizamos un estudio observacional retrospectivo (1987-2011) en 114 pacientes diagnosticados de NMPC y análisis de detección de la mutación V617F: 39 policitemias vera (PV), 71 trombocitemias esenciales y 6 mielofibrosis primarias. El porcentaje de alelos mutados fue evaluado en polimorfonucleares de sangre periférica mediante la técnica quantitative real time polymerase chain reaction (qRT-PCR, «reacción en cadena de la polimerasa cuantitativa en tiempo real»). En función de si la carga tumoral se encuentra entre 1-50% o es>50% los pacientes fueron diagnosticados de JAK2mut heterocigoto u homocigoto, respectivamente. Resultados: Se realizó el análisis cuantitativo por qRT-PCR en los 114 pacientes incluidos en el estudio, detectando la mutación V617F en 69 casos y siendo negativa para los 45 pacientes restantes. Encontramos que la presencia de mutación se asocia con la trombosis, y especialmente con la trombosis arterial. Además, y en la serie completa, la carga alélica homocigótica se asocia con diagnóstico de PV, edad avanzada, leucocitosis, mayor hematopoyesis y esplenomegalia. Conclusiones: La detección de la mutación V617F está asociada a una mayor incidencia de trombosis, leucocitosis y esplenomegalia. Su identificación nos permitiría una mejor estratificación pronóstica de los pacientes diagnosticados de NMPC (AU)

Background and objectives: Our study has investigated the presence of the mutation V617F in the JAK2 gene in patients diagnosed with chronic myeloproliferative neoplasms (MPNs). Furthermore, we determined if JAK2 (V617F) allelic burden associates with a specific clinical phenotype and if its quantification can be used as a marker to predict outcome and complications in patients with MPNs.Patients and methods: A retrospective observational study was conducted from 1987 to 2011 in the Haematology Department of La Paz University Hospital. The allelic burden was measured in 114 patients diagnosed with MPNs: 39 polycythemia vera (PV) patients, 71 essential thrombocythaemia patients and 6 primary myelofibrosis patients. The quantitative real-time polymerase chain reaction (qRT-PCR) technology was used to determinate the percentage of mutated alleles in peripheral blood neutrophils. Patients were divided in 2 groups: heterozygous if the result was≤50% of the tested cells, and homozygous if it was positive in>50% of the cells. Results: Sixty-nine patients were positive for the JAK2 mutation and 45 were negative. Among those positive, the mutation was associated with arterial thrombosis. In addition, we demonstrate in the homozygous group that the V617F mutation is associated to PV, advanced age, leukocytosis, marked haematopoiesis and splenomegaly. Conclusions: The presence of V617F mutation is associated with a higher incidence of thrombosis, leukocytosis and splenomegaly. The identification of mutation on the JAK2 gene could help in a better definition of evolution and prognostic stratification of the myeloproliferative disorders (AU)

[Clinical significance of the quantification of JAK2V617F allele burden in classical Ph-negative myeloproliferative neoplasms]. / Impacto clínico de la cuantificación de la carga mutacional de JAK2V617F en pacientes con neoplasias mieloproliferativas crónicas Ph negativas clásicas.

BACKGROUND AND OBJECTIVES: Our study has investigated the presence of the mutation V617F in the JAK2 gene in patients diagnosed with chronic myeloproliferative neoplasms (MPNs). Furthermore, we determined if JAK2 (V617F) allelic burden associates with a specific clinical phenotype and if its quantification can be used as a marker to predict outcome and complications in patients with MPNs. PATIENTS AND METHODS: A retrospective observational study was conducted from 1987 to 2011 in the Haematology Department of La Paz University Hospital. The allelic burden was measured in 114 patients diagnosed with MPNs: 39 polycythemia vera (PV) patients, 71 essential thrombocythaemia patients and 6 primary myelofibrosis patients. The quantitative real-time polymerase chain reaction (qRT-PCR) technology was used to determinate the percentage of mutated alleles in peripheral blood neutrophils. Patients were divided in 2 groups: heterozygous if the result was≤50% of the tested cells, and homozygous if it was positive in>50% of the cells. RESULTS: Sixty-nine patients were positive for the JAK2 mutation and 45 were negative. Among those positive, the mutation was associated with arterial thrombosis. In addition, we demonstrate in the homozygous group that the V617F mutation is associated to PV, advanced age, leukocytosis, marked haematopoiesis and splenomegaly. CONCLUSIONS: The presence of V617F mutation is associated with a higher incidence of thrombosis, leukocytosis and splenomegaly. The identification of mutation on the JAK2 gene could help in a better definition of evolution and prognostic stratification of the myeloproliferative disorders.

Platelet soluble CD40L and matrix metalloproteinase 9 activity are proinflammatory mediators in Behçet disease patients.

Platelets are the major source of plasma-soluble CD40L (sCD40L), an important inflammatory mediator. This study explored the impact of platelet-derived sCD40L on Behçet disease (BD), an autoinflammatory vasculitis. We also searched for influences by platelet matrix metalloproteinases (MMP) -2 and MMP-9, implicated in several inflammatory diseases, on CD40L shedding from platelet membrane. Platelet activation were studied by flow cytometry and aggregometry, surface expression of CD40L and platelet-leukocyte aggregates by flow cytometry, sCD40L by ELISA, cellular CD40L and CD40 levels by Western blot and MMPs activity by gelatin zymography. The effect of sCD40L on MMP9 expression was studied in cultured MEG-01 cells. Plasma and platelet-released sCD40L levels were higher in BD patients. No differences in platelet activation and in platelet-leukocyte aggregates formation were observed between BD patients and controls. Plasma and platelet MMP-9 levels were increased in BD patients, whereas there was no difference in platelet MMP-2 activity. Since a correlation between plasma sCD40L and platelet MMP-9 activity was observed, we studied the influence of sCD40L on MMP-9 levels in the megakaryoblastic cell line MEG-01. Treatment of MEG-01 cells with recombinant sCD40L increased MMP-9 but did not change MMP-2 levels. In conclusion, sCD40L release from platelets was mediated by MMP-9, and MMP-9 expression was in turn upregulated by sCD40L in the MEG-01 cell line. We conclude that platelets and megakaryocytes might participate in a positive feedback loop occurring between sCD40L and MMP-9 which would contribute to the proinflammatory state observed in BD.