Background: Microparticles (MPs) are small vesicles (0.1-1 micron) deriving from plasma membrane budding during homeostasis and cell activation. MPs express antigens and contain constituents from cell of origin and are increased in conditions that are characterized by high cell turnover or death, particularly inflammatory, autoimmune and neoplastic diseases. Myelofibrosis (MF) is a clonal neoplasia of the hematopoietic stem/progenitor cells characterized by distinctive abnormalities in megakaryocyte (MKC) development and platelet (PLT) activation. Mutations in 3 genes (JAK2, CALR, MPL) and chronic inflammation are the main pathogenetic drivers of MF. Ruxolitinib (RUX), a JAK1/2 inhibitor, suppresses both clonal myeloproliferation and release of proinflammatory cytokines, reducing splenomegaly and constitutional symptoms in around 50% of patients (pts). We hypothesized that MPs, as mediators of inflammation, could be overexpressed in MF and possibly predict responses to RUX. Aims: This study aims to: 1) enumerate circulating MK and PLT-derived MPs of MF pts; 2) evaluate the effect of RUX on MPs production by PLT and MK; 3) investigate whether circulating MK and PLT- MPs may be a biomarker of response to RUX. Methods: EDTA-anticoagulated peripheral blood from healthy donors (HD, n=10) and JAK2V617F positive MF pts (n=12) at intermediate-2/high IPSS risk was collected at baseline and 3 and 6 months after RUX therapy and immediately centrifuged. PLT (CD61+CD62P+) and MK (CD61+CD62P-)-derived MPs were analysed in PLT poor plasma samples by flow cytometry (CytoFLEX, Flow Cytometer-Beckman Coulter). The instrument was calibrated with MEGAMIX Beads (Beckman Coulter) with various diameters to cover the MPs (0.5 and 0.9 μm). Results: At 3 and 6 months, 5 out of 12 pts achieved a spleen response (R) according to 2013-IWG-MRT criteria. At baseline, the mean percentage of MK-derived MPs was significantly decreased (29±6 vs 72±5; p<0.0001) while that of PLT-derived MPs significantly increased (49±7 vs 11±1; p<0.0001) in MF pts compared to HD. However, the mean percentage of MK-derived MPs from pts not achieving a spleen response (NR) was significantly decreased compared to R (17±6 vs 45±5; p<0.005) and HD (17±6 vs 72±5; p<0.0001). By contrast, the mean percentage of PLT-derived MPs was significantly increased in NR compared to R (64±7 vs 37±9; p<0.05) and HD (64±7 vs 11±1; p<0.001). Of note, NR pts had significantly lower PLT number as compared with R pts (220±29 vs 422±98 p<0.05). No correlation was observed at baseline between the percentages of MK/PLT-derived MPs and platelet number, allele burden, splenomegaly and constitutional symptoms. At 3 and 6 months, RUX did not significantly modify the mean percentages of MK- and PLT-derived MPs compared to baseline values. Summary/Conclusion: At variance with HD, the majority of circulating MPs in JAK2V627F mutated MF pts at intermediate-2/high IPSS risk derived from PLTs. RUX therapy did not modify the MK/PLT-derived MPs pattern, suggesting that JAK1/2 inhibition does not seem to affect the pathways of MK/PLT MPs production or clearance. Most importantly, MPs evaluation at baseline is significantly associated with subsequent spleen response. Specifically, NR pts had increased percentages of PLT-derived MPs with a concomitant reduction of PLT number. This could be related to a state of PLT hyper-activation with hyper-production of MPs. Further studies are needed to confirm whether MPs may actually be considered a biomarker of disease activity and response to RUX.

CIRCULATING PLATELET AND MEGAKARYOCYTE-DERIVED MICROPARTICLES OF JAK2V617F MUTATED PATIENTS WITH MYELOFIBROSIS ARE DISREGULATED: A NOVEL LIQUID BIOPSY TOOL OF RESPONSE TO RUXOLITINIB?

BARONE, MARTINA;FORTE, DORIAN;Daria Sollazzo;Marco Romano;Marco Spinsanti;Emanuela Ottaviani;Michele Cavo;Nicola Vianelli;Francesca Palandri;Lucia Catani
2017

Abstract

Background: Microparticles (MPs) are small vesicles (0.1-1 micron) deriving from plasma membrane budding during homeostasis and cell activation. MPs express antigens and contain constituents from cell of origin and are increased in conditions that are characterized by high cell turnover or death, particularly inflammatory, autoimmune and neoplastic diseases. Myelofibrosis (MF) is a clonal neoplasia of the hematopoietic stem/progenitor cells characterized by distinctive abnormalities in megakaryocyte (MKC) development and platelet (PLT) activation. Mutations in 3 genes (JAK2, CALR, MPL) and chronic inflammation are the main pathogenetic drivers of MF. Ruxolitinib (RUX), a JAK1/2 inhibitor, suppresses both clonal myeloproliferation and release of proinflammatory cytokines, reducing splenomegaly and constitutional symptoms in around 50% of patients (pts). We hypothesized that MPs, as mediators of inflammation, could be overexpressed in MF and possibly predict responses to RUX. Aims: This study aims to: 1) enumerate circulating MK and PLT-derived MPs of MF pts; 2) evaluate the effect of RUX on MPs production by PLT and MK; 3) investigate whether circulating MK and PLT- MPs may be a biomarker of response to RUX. Methods: EDTA-anticoagulated peripheral blood from healthy donors (HD, n=10) and JAK2V617F positive MF pts (n=12) at intermediate-2/high IPSS risk was collected at baseline and 3 and 6 months after RUX therapy and immediately centrifuged. PLT (CD61+CD62P+) and MK (CD61+CD62P-)-derived MPs were analysed in PLT poor plasma samples by flow cytometry (CytoFLEX, Flow Cytometer-Beckman Coulter). The instrument was calibrated with MEGAMIX Beads (Beckman Coulter) with various diameters to cover the MPs (0.5 and 0.9 μm). Results: At 3 and 6 months, 5 out of 12 pts achieved a spleen response (R) according to 2013-IWG-MRT criteria. At baseline, the mean percentage of MK-derived MPs was significantly decreased (29±6 vs 72±5; p<0.0001) while that of PLT-derived MPs significantly increased (49±7 vs 11±1; p<0.0001) in MF pts compared to HD. However, the mean percentage of MK-derived MPs from pts not achieving a spleen response (NR) was significantly decreased compared to R (17±6 vs 45±5; p<0.005) and HD (17±6 vs 72±5; p<0.0001). By contrast, the mean percentage of PLT-derived MPs was significantly increased in NR compared to R (64±7 vs 37±9; p<0.05) and HD (64±7 vs 11±1; p<0.001). Of note, NR pts had significantly lower PLT number as compared with R pts (220±29 vs 422±98 p<0.05). No correlation was observed at baseline between the percentages of MK/PLT-derived MPs and platelet number, allele burden, splenomegaly and constitutional symptoms. At 3 and 6 months, RUX did not significantly modify the mean percentages of MK- and PLT-derived MPs compared to baseline values. Summary/Conclusion: At variance with HD, the majority of circulating MPs in JAK2V627F mutated MF pts at intermediate-2/high IPSS risk derived from PLTs. RUX therapy did not modify the MK/PLT-derived MPs pattern, suggesting that JAK1/2 inhibition does not seem to affect the pathways of MK/PLT MPs production or clearance. Most importantly, MPs evaluation at baseline is significantly associated with subsequent spleen response. Specifically, NR pts had increased percentages of PLT-derived MPs with a concomitant reduction of PLT number. This could be related to a state of PLT hyper-activation with hyper-production of MPs. Further studies are needed to confirm whether MPs may actually be considered a biomarker of disease activity and response to RUX.
haematologica Journal of the European Hematology Association Owned & published by the Ferrata Storti Foundation
Martina Barone, Francesca Ricci, Dorian Forte, Daria Sollazzo, Marco Romano, Marco Spinsanti, Emanuela Ottaviani, Michele Cavo, Nicola Vianelli, Pierluigi Tazzari, Francesca Palandri, Lucia Catani
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/629105
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