Immunophenotypic, Cytogenetic and Functional Characterization of Circulating Endothelial Cells in Myelodysplastic Syndromes.
Authors: Della Porta Matteo G. , Malcovati Luca , Rigolin Gian Matteo , Rosti Vittorio , Travaglino Erica , Boveri Emanuela , Galli Anna , Boggi Sabrina , Invernizzi Rosangela , Cazzola Mario 
Published: April 2020

Abstract
Tumor growth and dissemination require active endothelial proliferation, a process referred to as neo-angiogenesis. Tumor cells can elicit the sprouting of new blood vessels from pre-existing capillaries by the active release of angiogenic factors. There is growing evidence that bone-marrow derived endothelial cells may contribute to this process, and circulating endothelial cells (CEC) have been identified that are mobilized from the bone marrow and transported through the blood-stream to become incorporated into the vascular bed. In myelodysplastic syndromes (MDS) the expansion of the dysplastic clone is a multi-step process favored by perturbed interactions between hematopoietic precursors and marrow stromal microenvironment. In the present study, circulating endothelial cells were analyzed by flow cytometry and correlated with clinical parameters as well as with bone marrow vasculature in a cohort of 128 MDS patients followed at the Division of Hematology, University of Pavia & IRCCS Policlinico San Matteo, Pavia, Italy. Endothelial cells were identified as CD146+/CD34+/CD45− cells; in addition, the capability of these cells to produce endothelial colonies was tested in vitro. In order to clarify whether they were tumor-derived, CEC were isolated and analyzed by fluorescence in situ hybridization (FISH). A significantly higher number of CEC was observed in MDS patients compared with healthy controls (P&lt;.001). An effect of WHO category on CEC level was noticed (P=0.01), patients with low-risk MDS (RA, RARS and MDS with del5q) presenting the highest values. A negative correlation was found between CEC number and IPSS-risk group (r=−0.55, P&lt;.001): the higher the IPSS risk, the lower the CEC level. Moreover, a negative correlation was noticed between CEC level and duration of the disease (r=−0.69, P&lt;0.001). We calculated a progenitor-to-mature endothelial cell ratio (CEC ratio) on the basis of the CD133 marker, which is gradually lost during endothelial differentiation. The CEC ratio was increased in MDS patients in comparison with healthy subjects (P&lt;0.001), the highest CEC ratio being found at diagnosis with respect to the more advanced phases of the disease (P&lt;0.001). A positive correlation was observed between microvessel density and CEC in MDS (r=0.44, P=0.001), low-risk patients showing the strongest association (r=0.72, P&lt;0.001). We then tested the capability of peripheral blood mononuclear cells to produce endothelial colonies in vitro, and found that MDS patients presented a significantly reduced capability to produce CFU-End with respect to healthy subjects. Finally, circulating endothelial cells were isolated in 5 MDS patients with abnormal karyotype. FISH analysis showed that in MDS patients a significant proportion of CEC was tumor-derived since they carried the chromosomal aberrations detected in the neoplastic clone (range 39.3%–84%). In summary, we found that patients with MDS have significantly higher levels of CEC compared with healthy controls. CEC are mainly increased in early stages of the disease and tend to decrease in the more advanced phases. We also demonstrated that CEC and myeloid cells share the same chromosomal abnormalities suggesting a close relationship between myelodysplastic clone and endothelial compartment.