Myelofibrosis (MF) is a clonal neoplasia of the hematopoietic stem/progenitor cells characterized by distinctive abnormalities in megakaryocyte (MK) development and platelet (PLT) activation. Therapy with Ruxolitinib (RUX), a JAK1/2 inhibitor, suppresses both clonal myeloproliferation and release of proinflammatory cytokines, reducing splenomegaly and constitutional symptoms in around half of patients (pts). However, a deeply understanding of the activated inflammatory pathways and the biological effects of RUX is still lacking. Microparticles (MPs) produced by cells may occur as a key cellular communication process in homeostasis, which may increase under inflammatory stimuli and neoplastic diseases. Most of circulating MPs are of PLT and MK origin. Their role and modulation in the inflammatory microenvironment of MF are elusive.

We evaluated whether circulating MK and PLT-derived MPs are dysregulated in MF and how the inflammatory microenvironment may drive their pattern. Also, we investigated whether circulating MK and PLT-derived MPs may be considered as a novel biomarker of response to RUX.

EDTA-anticoagulated peripheral blood (PB) was collected from 42 MF pts, 13 Polycythemia Vera (PV)/Essential Thrombocythemia (ET) pts and 10 age/sex-matched healthy donors (HD). Samples from MF, ET/PV pts were obtained at diagnosis or out of cytotoxic treatment for at least 3 months; 14 MF pts were studied also after 6 months of RUX therapy. Spleen response (SR) was evaluated according to the 2013 IWG-MRT criteria. PLT (CD61+CD62P+) and MK (CD61+CD62P-)-derived MPs were analysed in PLT poor plasma samples by flow cytometry (CytoFLEX, Beckman Coulter, MPs ranging 0.5-0.9 μm).

MF pts were JAK2V617F (n=24), CALR (n=9) and MPL (n=4) mutated; 5 pts were TN . The mean percentage of PLT-derived MPs of JAK2V617F mutated pts was significantly increased as compared with HD (p<0.05; Fig. 1A). On the contrary, as a sort of compensatory mechanism, the mean percentages of MK-derived MPs were significantly decreased in JAK2V617Fand TN pts as compared to HD (p<0.05, respectively; Fig. 1B). Interestingly, among MPNs, PLT-derived MPs were significantly increased in ET pts only as compared with HD (p<0.05).

MF pts were at low/intm-1 (n=16), or at intm-2/high IPSS score (n=26). Higher-risk (HR)- JAK2V617F pts only showed a significantly increase of PLT-derived MPs along with a significant decrease in the percentage of MK-derived MPs as compared to lower risk (LR)- JAK2V617F and HD (p<0,0001, respectively; Fig. 1C-D).

Remarkably, at baseline, contrary to pts with no response (NR; n=7), pts achieving a SR (R; n=7) showed a low PLT-/MK-derived MPs ratio (Fig. E, baseline), as result of the reduction of MK-derived MPs in NR pts (p<0,05) (Fig. 1G). Accordingly, it may prompt to consider this ratio as potential predictive marker to RUX response. After 6 months of RUX, the PLT-/MK-derived MPs ratio of R pts was significantly decreased as compared with that of NR pts (p<0,01) (Fig. 1E, 6-months post-RUX); this is mainly due to the strong reduction of PLT-derived MPs in R pts (p<0,001) (Fig. 1F).

To evaluate whether inflammatory status may play as a key regulator of MPs, we correlated the level of crucial inflammatory factors (Interferon (IFN)-γ, Interleukin (IL)-1β, IL-6, IL-8, Tumor Necrosis factor (TNF)-α, Thrombopoietin (TPO)) to circulating MPs. Surprisingly, we reported a highly inverse relationship between levels of IFN-γ (r=-0,5534; p<0,01), IL-1β (r=-0,4648; p<0,01), TNF-α (r=-0,4444; p<0,05) and MK-derived MPs percentages, highlighting an impaired response to inflammation. Concomitantly, we observed a positive correlation between IL8 with MK-derived MPs hinted at its implication in MK dysregulation. The percentages of PLT-derived MPs were positively correlated with only TPO plasma levels (r=0.4299; p<0.05), that may influence PLT/MK-derived MPs ratio by activating PLT.

Overall our results demonstrate that in MF pts the inflammatory status may contribute to drive MK and PLT-derived MPs dysregulation. We also show that the mutation status impacts the proportion of circulating MK-and PLT-derived MPs, suggesting a role of mutated JAK2 in MPs biogenesis/clearance and, ultimately, in cell (MK, PLT) to cell communication. Interestingly, MK vs PLT-derived MPs pattern may be a biomarker of disease identifying JAK2V617F -mutated pts at high IPSS risk and RUX responsive.

DF, MB, FR and FP, LC equally contributed

Disclosures

Martinelli: Ariad/Incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy.

Author notes

*

Asterisk with author names denotes non-ASH members.

Sign in via your Institution