Abstract
INTRODUCTION: Extracellular vesicles (EVs), including exosomes, shedding vesicles (microvesicles: MVs) and apoptotic bodies, are membrane vesicles of 40-3,000 nm that are released from many cell types, such as red blood cells, platelets, lymphocytes, dendritic cells, endothelial cells and tumour cells. They have a key role not only in the regulation of normal physiological processes, but also in the pathology underlying several diseases. Recently, it was discovered that EVs circulate in bodily fluids of cancer patients and could serve as potential diagnostic biomarkers. However, the presence and role of peripheral circulating EVs in hematological malignancies (HMs) remain unclear. The aim of this study was to investigate EVs in peripheral blood of HM patients, defining the EVs count, size and phenotype.
METHODS: Peripheral blood samples were obtained prior to treatment from 87 subjects: 6 Chronic Lymphocytic Leukemia (CLL), 10 Non-Hodgkin's Lymphoma (NHL; 5 follicular, 5 diffuse large B-cell), 6 Waldenstrom's Macroglobulinemia (WM), 6 Hodgkin's Lymphoma (HL), 6 Multiple Myeloma (MM), 5 Acute Myeloid Lukemia (AML), 19 Myeloproliferative Neoplasms [(MPNs; 5 Essential Thrombocythemia (ET), 5 Polycythemia Vera (PV), 5 Primary Myelofibrosis (PMF), 4 Chronic Myelogenous Leukemia (CML)], 5 Myelodysplastic Syndrome (MDS) and 28 healthy controls. EVs were isolated from serum of peripheral blood by ultracentrifugation steps. For calculation of counts, TruCOUNT beads were added immediately prior to analysis by flow cytometry (FACS). For size assessment, Megamix beads with specific size (0.3,0.9, 3.0 micrometer) were used. For phenotype characterization, a panel of specific antibodies (anti CD59, CD61, CD62E, CD19, CD20, CD38, CD138, CD13, CD30) were employed in a FACS analysis.
RESULTS: MVs count was significantly higher in patients with CLL, WM, HL, MM, AML and CML (median values 890, 3227, 1218, 914, 656 and 1275 MVs/microliter of serum, respectively) compared to healthy controls (median value 270 MVs/ microliter of serum, ***P<0.001). MV levels in the other HMs, although sligthly higher when compared to healthy controls, did not mantain statistical significance when p value was corrected for the number of malignancies analysed. (Figure)
In CLL and MDS, through the number of cases was small, MVs counts directly correlated with Rai stage and R-IPSS risk, respectively.
As regards vesicles volume, all HMs generated a distinct population of MVs with smaller size when compared with controls. As expected, MVs were consistently Annexin V positive and expressed a common membrane protein, CD59, an ubiquitous complement regulator. Part of MVs originated from platelets (median value 400 CD61 positive/ microliter of serum) and endothelial cells (median value 60 CD62E positive/ microliter of serum).
Interestingly, MV phenotype was disease specific: a relevant amount of MVs expressed surface specific proteins related to malignancy. In particular, we found MVs positive for CD19 and/or CD20 in CLL, WM and NHL, CD38 and/or CD138 in MM, CD13 in AML, MPNs and MDS, CD30 in HL. These markers were not significantly expressed on the surface of healthy subject MVs.
CONCLUSIONS: In this study, for the first time, serum MVs in a panel of HMs were analysed. We found that patients with various types of HMs release elevated peripheral blood MV levels selectively displaying markers of the underlying malignancy. MVs could be therefore useful as potential novel biomarkers in HMs.
No relevant conflicts of interest to declare.
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