Abstract
Background:Myeloproliferative neoplasms (MPN) are chronic myeloid cancers characterized by the overproduction of mature blood cells and a tendency to evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro‐phagocytic signal and is counteracted by concomitant expression of anti‐ phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood.Aims:Investigate the expression and cellular localisation of CALR and CD47 in untreated and treated patients with essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF), in comparison with healthy controls.Methods:Mononuclear cells were collected by Ficoll separation, from peripheral blood of 27 MPN patients (8 PV, 16 ET, 3 MF), and from 4 controls. In total 14 patients received cyto‐reductive therapies (Hydroxyurea, Anagrelide and Ruxolitinib). Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting.Results:Total CALR and CD47 protein expression increased in MPN samples comparing with controls (CALR‐ 7.9 vs 5.1; CD47‐ 2.7 vs 2.2 fold, respectively). CD47 showed higher expression of its overall protein on MPN cells membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR expression in all MPN subtypes when patients are treated with cyto‐reductive agents (ET‐ untreated 43.3% vs treated 2%, PV‐ 3.6% vs 2.2%, ET‐ 21% vs 11%). Interestingly we have observed a significant increase in CD47 expression after treatment in MF and PV cell membranes (CD47 in MF‐ untreated 11.8% vs treated 34.3%, PV‐11.4% vs 35.9%), suggesting an anti‐phagocytic effect induced by cytotoxic drugs in MF and PV cells. In ET cell membranes however, CD47 expression is reduced after cyto‐reductive treatment (22% vs 16.6%), suggesting instead a pro‐phagocytic effect.Summary/Conclusion:CD47, but not CALR, is overexpressed on the membrane of patients with MPN. This opposes previous studies in solid tumours, which showed significant increases of both CALR and CD47, suggesting a role for CD47 as a strong anti‐phagocytic signal responsible for immune survival in MPN. All MPN patients decreased CALR expression during cyto‐reduction therapy, but we observed a significant difference in CD47 expression across different MPN subtypes. In fact, treating MF and PV patients with cyto‐reductive agents, increases CD47 expression leading to a stronger anti‐phagocytic signal, while in ET we observed a reduction, suggesting a more pro‐phagocytic reaction to cytotoxic drugs in ET. These preliminary data suggest how the interaction between the immune system and MPN cells can be altered by cytotoxic drugs differently according to MPN sub‐type. A larger cohort of patients is required to confirm those data and allow a further stratification based of type of cyto‐toxic drugs. The use of anti‐CD47 antibodies could represent a new strategy to enhance the pro‐phagocytic signal via increasing the CALR expression, and in combination with standard cyto‐reduction therapy, might represent a new therapeutical strategy in MPN.
Published Version
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