Expression of Cytoplasmic and Surface VEGF by Plasma Cells in Myeloma and Related Disorders.

Background: Previous studies quantitating VEGF in plasma cells from multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and smoldering myeloma (SMM) have found no significant difference in expression between the groups. These studies have been done using immunohistochemistry, quantitative RT-PCR, and Western Blots using material from isolated CD138+ plasma cells. These studies may have been limited by specificity of reagents, low numbers of plasma cells, or heterogeneity within the plasma cell population. Asosingh et al have shown that 5T2MM murine myeloma cells show greater VEGF expression in the CD45- compared to the CD45+ plasma cell compartment (Asosingh K, Blood2004;103:3131–7). Quantitative flow cytometric methods allow for the quantitation of antigen expression with high sensitivity and allow discrimination among cell subsets. Flow studies have suggested that myeloma patients have both CD45+ and CD45 - plasma cell subsets and that each compartment may contain phenotypic and functionally different types of clonal plasma cells. This two part study evaluated bone marrow plasma cells for intracellular and surface levels of VEGF in MM, MGUS, SMM, amyloid, and normal controls. Methods: Cytoplasmic VEGF expression (cVEGF) was measured on lysed and permeabilized (FACS TM Permeabilizing Solution, BD Biosciences, San Jose, CA) whole bone marrow samples using anti-VEGF PE (R and D Systems, Minneapolis, MN). With each run, Quantum TM Simply Cellular Beads (Bangs Laboratories, Fishers, IN) were stained with the same VEGF antibody to create a standard curve for calculation of MESF/ABC (antibody binding capacity) values. A median VEGF MESF value was calculated for the plasma cells, lymphocytes, and granulocytes from each patient sample. Surface expression of VEGF (sVEGF) was determined using CD138 FITC/ VEGF PE/ CD45 Percp/ CD38 APC staining on ACK lysed whole bone marrow cells. sVEGF expression was determined on the plasma cell population as a whole and on the individual CD45 fractions. Median expression and proportion of cases with plasma cells expressing >20% sVEGF were calculated. Cytoplasmic kappa and lambda staining was also done to determine clonality. Results: cVEGF MESF differences within the plasma cell populations were greater than in the other cell types. The MESF was higher in MM (n=14 patients, median MESF 247,866) compared to MGUS/SMM (n=14, median MESF 89,794) and amyloid (n=5, median MESF 134,429)(p=0.038). The lymphocytes and granulocytes in each group had similar MESF values, but the median MESF was lower than in the plasma cells (median 41,485 for lymphocytes, and 24,227 for granulocytes). sVEGF was expressed by less than 20% of plasma cells in all groups studied. Similar results were also seen in the CD45- plasma cell fraction. In the CD45+ plasma cell fraction, all groups showed varying levels of positive expression: MM (n=15 patients; median, 52% cells positive), normal controls (n=4; 51%), MGUS/SMM (n=11; 34%). Conclusions: cVEGF expression was significantly higher in MM compared to SMM/MGUS in this study. On the other hand, sVEGF expression was confined to the CD45+ plasma cell compartment in all groups studied, with a trend to higher % positive cells in the MM group compared to MGUS. We are working on studying levels of secreted VEGF in various human plasma cell subsets, both alone and in conjunction with stromal cell contact.