PB2386 BEHAVIOUR OF CIRCULATING ENDOTHELIAL CELLS IN AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PATIENTS WITH MULTIPLE MYELOMA
Authors: O. Annibali, C. Gregorj, V. Tomarchio, M. Di Cerbo, L. Antonelli, D. Armiento, A. Scardocci, M. Tafuri, E. Circhetta, C. Sarlo, B. Vincenzi, M. C. Tirindelli, G. Avvisati
Journal: HemaSphere (2019)

Background: Neoangiogenesis has a crucial role in the pathogenesis and progression of hematologic malignancies, in particular in Multiple Myeloma (MM). Circulating Endothelial Cells (CECs) are a subtype of stem cell capable to contribute to neovascularization by inducing tumor progression and metastasis and by repairing damage to bone marrow vasculature after stem cell transplantation. Different flow cytometry methods for CECs characterization have been published so far, none of them have reached consistent conclusions. However, recently, utilizing a highly optimized method based on a polychromatic flow cytometry Lyotube (BD), we proved in a national multicenter study the possibility to reach a high level standardization in CEC count and analysis. Aims: The aim of our study was to assess the kinetics of CECs in patients with MM undergoing autologous hematopoietic stem cell transplantation (ASCT). Methods: Samples were collected in three EDTA tubes and for each sample, 20 × 106 leukocytes were processed as already described (Lanuti 2016 e 2017). In brief, cells, after a erythrocyte-lysis step, were added to the lyophilized cocktail (Lyotube BD) of reagents plus 1 μM Syto16 (Thermo Fisher Scientific, Eisai, Medipost - US). Samples were incubated in the dark for 30 minutes at 4 °C, and re-suspended in 1.5 mL of FACSFlow (BD Biosciences). 1-2 × 106 events/sample with lymph-monocyte morphology were acquired by flow cytometry (FACSCanto II- BD). A threshold combination was used on Forward Scatter (FSC) and Fluorescein Isothiocyanate (FITC-Syto16) channels to get rid of very small and non-nucleated events. Compensations were calculated using CS&T bright beads (BD Biosciences). CEC were identified, as 7-ADDneg/Syto16pos/CD45neg/CD34pos/CD146pos. Venous blood samples for CECs analysis were collected at different time points: before chemotherapy (T0), before HSC, on the day of HSC infusion (T1), 24 h after HSC transplantation (T2), on the day of neutrophil engraftment (T3), at 100 days after transplantation (T4). Results: MM (#26) patients responding to standard treatments were enrolled in the study.: 11/26 (42%) were females and 15/26 (58%) were males with a median age of 59 years. All patients received high dose chemotherapy followed by ASCT. The median CECs number at T0 was 78.0/ml, vs normal value < 30/ml (obtained in 50 blood donors). Considering the kinetics of CECs we observed a costant increase of CECs from T0 to T3 followed by decrease at T4 (figure 1). Using the median value of CECs at T3 we were able to divide our population in 2 groups > or ≤ 619 /ml. By this cut off, patients with more infective complications had a CECs value > 619/ml (9/13 vs 2/13; P = .005).Summary/Conclusion: This study suggest direct evidence that CECs value may be a function of endothelial damage caused by conditioning regimen as demonstrated by the increase of their level during engraftment period. A more severe endothelial damage is reflected by the increase of infective complications in patients with higher CECs value at T3. Further studies on larger population are needed to confirm these preliminary results.