Multi-Parameter Flow Cytometry(FC) Can Detect Proliferating Myeloma Cells After Treatment: Implications for Targeting the Myeloma Proliferative Component

Multiple myeloma results from an accumulation of monoclonal nonproliferating plasma cells arising from a small subpopulation of proliferating myeloma cells. In an effort to optimize detection of the human myeloma cell growth fraction and obviate the need for slide review, a novel FC strategy was developed combining elements of light chain restriction, surface antigen expression, and ploidy analysis.

Bone marrow cells from 41 patients with plasma cell proliferative disorders were lysed with ACK and resuspended in 3% BSA. Cells were stained using a 6-color assay with anti-CD45, anti-CD38, anti-CD138, and anti-CD19. Cells were washed and 100 ul Caltag solution A was added for 15 min. Cells were washed and 100 ul of Caltag solution B, anti-kappa and anti-lambda were added for 10 min. Cells were washed and 100 ul PBS and 3 ul RNAse are added for 15 min. Cells were washed and 400 ul of a 1:1000 solution of 3uM DAPI in Tris 0.1% NP-40 was added. Cells were incubated at 4°C for 45 minutes before running on a FACSCanto instrument for ploidy determination. All patients were analyzed both by flow cytometry and the slide based plasma cell labeling index. Results: Forty-one patients were studied; 34 demonstrated a proliferative fraction and 7 had too few plasma cells for analysis after therapy. Of the 34 patients, 6 had MGUS/SMM, 5 newly diagnosed MM, 7 amyloid, and 16 were treated MM. The mean percent proliferating cells were 1.1% (range 0 – 8.6%) with PCLI and 1.4% (range 0.1 – 12.7%) by flow. The correlation between PCLI and flow gave a RSquare value of 0.54. Twelve patients with a PCLI of 0% had a flow proliferation between 0.1 – 1.4% (mean 0.49%). Treated patients received lenalidomide, dexamethasone, bortezomib, and/or autologous transplantation. All 16 treated myeloma patients with adequate plasma cells had a flow proliferation between 0.2 – 12.7% (mean 2.2%). Conclusion: Flow cytometry offers a useful way to detect the proliferative myeloma component at diagnosis and after treatment. The continued presence of proliferating myeloma cells after treatment may explain why most patients relapse and offers another important marker to monitor and cell population to target in patients with active disease.

Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.