PF539 MIR‐223 IS SUPPRESSED IN EXOSOMES OF PATIENTS WITH HIGH‐RISK MYELODYSPLASTIC SYNDROMES AND MAY BE A MARKER OF RESIDUAL GRANULOCYTIC‐MONOCYTIC DIFFERENTIATION
Authors: B. Benites, A. Duarte, M. Alvarez, I. Santos, L. Ribeiro, E. de Paula, S. Saad
Journal: HemaSphere (2019)

Background: Quantification of exosomal miRNAs in body fluids represents a prominent source of biomarkers for a variety of conditions. Several miRNAs have been described as potential predictors of severity and progression in neoplastic diseases, pointing to a class of less invasive and readily accessible prognostic tools. Among them, mir‐223 has been studied with contradictory findings: mir‐223 has been shown to play an important role in quimiorresistence in solid tumors (Cheng Q, et al., Mol Med Rep. 2017;15:597–604); however, suppression of mir‐223 in Acute Myeloid Leukemia (AML) is associated with lack of differentiation and adverse cytogenetic profile (Akhter A, et al., Appl Immunohistochem Mol Morphol, 2015;23:733–9). Aims: The aim of this study was to evaluate the expression of mir‐223 in exosomes extracted from serum of patients with myelodysplastic syndromes (MDS), AML and healthy blood donors and to explore the role of mir‐223 in the differentiation of human hematopoietic stem cells. Methods: Samples of venous blood were obtained from 14 patients with AML, 28 patients with MDS (prior to any treatment), and 19 healthy controls. Exosomes were isolated using the ExoQuick ® precipitation solution; their concentration and size distribution were characterized by Nanoparticle Tracking Analysis (NanoSight, UK). Exosomal RNA was prepared using miRNeasy Micro Kit and submitted to reverse transcription with miScript II RT kit (QIAGEN, USA) and expression levels of mir‐223 and mir‐192 (endogenous control) were assessed by RT‐PCR using specific primers. CD34+ progenitors were purified from human umbilical cord blood using positive immunomagnetic selection and expanded in culture medium supplemented with G‐CSF, IL‐3 and SCF. The expression levels of mir‐223 were analyzed in cultured cells on days 0, 7, and 14; expression of CD34, CD14 and CD15 was verified by flow cytometry and morphologic changes were observed by examining Wright‐Giemsa‐stained cytospin smears. Results: The concentration of exosomes in serum samples ranged from 5.6 x 10 11 to 3.3 x 10 13 particles per μl of liquid suspension, with significantly lower concentrations in the patient samples in relation to healthy individuals ( p = 0.004). A statistically significant decrease in the expression of mir‐223 was observed in exosomes from individuals with high‐risk MDS compared to healthy subjects ( p = 0.002) and to patients with low‐risk MDS ( p = 0.012). Interestingly, in the total group of patients, there was a statistically significant positive correlation between the expression of mir‐223 in the exosomes and countings of: total leukocytes (Spearman's r s = 0.37, p = 0.003), neutrophils ( r s = 0.392, p = 0.002), monocytes ( r s = 0.484, p &lt; .001), lymphocytes ( r s = 0.442, p &lt; .001) and platelets ( r s = 0.387, p = 0.002) in peripheral blood. During granulocytic‐monocytic differentiation of normal human CD34+ cells, we observed a 4‐fold increase in mir‐223 expression in cultured cells after 7 days, reaching almost 6‐fold increase after 14 days of culture, with granulocytic‐monocytic differentiation demonstrated by cytology and immunophenotyping with CD14 and CD15 markers. Summary/Conclusion: The results here obtained showed that levels of mir‐223 in peripheral blood exosomes are suppressed in patients with high‐risk MDS and correlate with cell counts in both MDS and AML. As mir‐223 levels progressively increase during normal granulocytic‐monocytic differentiation, we speculate that mir‐223 may represent a marker of residual differentiation in MDS and AML.