Higher Vascular Endothelial Growth Factor (VEGF) and Endothelial Progenitor Cells (EPCs) in Multiple Myeloma (MM) Patients (pts) as a Reflection of Their Governing Role in Pathological Angiogenesis: Comparison of VEGF and EPC Levels Between Healthy Donors (HD), MGUS and MM Pts and Correlation Analysis with MM Activity
Authors: Udi Josefina , Kleber Martina , Wider Dagmar , Wäsch Ralph , Engelhardt Monika 
Published: April 2020

Abstract
Introduction: In MM pathogenesis, angiogenesis and growth factors have a governing role. VEGF, secreted by malignant bone marrow (BM) plasma cells (PCs) and stroma, acts as an important mediator of tumor angiogenesis. VEGF has been suggested as an adverse prognostic factor, being elevated in advanced and plasmablastic MM. Circulating as well as BM-residing endothelial cells (ECs) have been shown to contribute to angiogenesis in MM as well as other tumors. Moreover, endothelial progenitor cells (EPC) have been demonstrated to contribute to vascular repair and to be decreased in number and function with end-stage renal impairment (RI). Whereas VEGF and/or EPCs have been described in small former analysis in MM, larger comparisons with MGUS pts and healthy donors (HD) are lacking, differences in various MM subsets (such as BM; peripheral blood and apheresis [AP] samples) have not been addressed, nor the role of EPCs and hemangioblasts in advanced vs. mild RI in MM pts.
Methods: We sought to characterize ECs (namely VEGF+ cells, EPCs as VEGFR2+/CD133+/CD34+, hemangioblasts as VEGF+/CD34+) and other subtypes (CD34+, CD45+, CD38+, CD45+/CD38+, CD45−/CD38+) via multiparametric flow cytometry. This was performed in MM pts (n=70), MGUS pts (n=8) and HD (n=14). In MM, BM (n=70), PB (n=14) and AP (n=21) specimens were compared, as well as changes in EPCs and hemangioblasts with and without mild or severe RI. Renal function was determined via estimated glomerular filtration rate (eGFR), classifying mild RI as eGFR &lt;90ml/min/1.73m2 and severe RI as eGFR &lt;30ml/min/1.73m2.
Results: MM pts’ age, BM-infiltration and serum creatinine were 63 years (range: 35–84), 15% (0–96) and 0.91mg/dl (0.5–8.6), respectively. BM specimens from MM and MGUS pts showed 4-fold and 2-fold higher VEGF levels, respectively as compared to HD (Table 1). EPCs were also elevated in MM as compared to MGUS and HD (Table 1). Hemangioblasts, CD45+/CD38+ and CD45−/CD38+ were increased in MM BM specimens as compared to MGUS and HD, whereas CD45+ and CD34+ cell numbers were decreased in myeloma specimens (Table 1).
Table 1. Median endothelial cells and other subtypes in MM, MGUS and healthy donors
BM MM (n=70) BM MGUS (n=8) BM healthy donors (n=14) VEGF+ (%) 0.38 (0 – 12.9) 0.18 (0 – 0.7) 0.09 (0 – 0.6) EPCs (%) 0.03 (0 – 0.4) 0.02 (0 – 0.07) 0.01 (0 – 0.2) VEGF+/CD34+ (%) 0.21 (0 – 2.2) 0.11 (0 – 0.4) 0.04 (0 – 0.5) CD34+ (%) 0.65 (0 – 6.6) 1.23 (0.02 – 4.0) 1.50 (0.07 – 2.8) CD45+ (%) 39.54 (2.8 – 99.1) 39.34 (13.8 – 69.7) 51.70 (10.3 – 90.9) CD45+/CD38+ (%) 24.61 (0.9 – 89.7) 21.68 (1.4 – 49.4) 17.20 (2.6 – 56.9) CD45−/CD38+ (%) 1.54 (0 – 77.7) 1.15 (0 – 2.4) 0.62 (0.1 – 5.6)
The comparison of BM, PB, AP specimens in MM showed similar VEGF levels in BM and PB with 0.38% which were increased in AP specimens with 0.5%. This was similarly observed for EPCs with 0.03% in BM and PB as compared to 0.04% in AP samples. Other markers showed similar values for CD34, CD45, CD38+ cells in BM and PB; similar hemangioblast numbers in all 3 subsets, and higher CD34+ and CD45+ cells, and lower CD45−/CD38+ cells in AP specimens. Correlation of EPCs and hemangioblasts with renal function revealed that EPCs decreased with RI, whereas hemangioblasts remained comparable (Table 2).
Table 2. Median EPCs and hemangioblasts (VEGF/CD34) with and without RI
EPCs (%) VEGF+/CD34+ (%) eGFR &gt;90 (n=41) 0.050 (0 – 0.41) 0.195 (0 – 0.86) eGFR &lt;90 (n=46) 0.025 (0 – 0.41) 0.230 (0 – 0.8) eGFR &gt;30 (n=81) 0.030 (0 – 0.41) 0.210 (0 – 2.23) eGFR &lt;30 (n=6) 0.025 (0 – 0.41) 0.190 (0 – 2.23)
Conclusions: These results demonstrate that all ECs, namely VEGF+ cells, EPCs and hemangioblasts are higher in MM than MGUS and HD. Lower CD34+ and CD45+ cells in MM suggest this as a result of the disease and most likely also due to anti-MM therapy. We observed differences in BM, PB and AP specimens in MM pts. RI influenced EC numbers. These results suggest that elevated ECs in MM may reflect disease activity and may be useful as MM biomarkers. The quantification of ECs in MM may also be informative to monitor the efficacy of anti-angiogenic treatment, such as thalidomide and lenalidomide. Further analyses will evaluate the prognostic significance of EPCs, hemangioblasts and other markers in MM, their role in mild and severe RI is ongoing, as well the correlation with disease outcome.