Detection of clonal plasma cells in POEMS syndrome using multiparameter flow cytometry

Abstract

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19^- in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome.

Keywords: EuroFlow; Immunophenotype; Multiparameter flow cytometry; POEMS syndrome.

Figure 1

Different gating strategies in MM-flow and POEMS-flow. Gating strategies in (A) MM-flow and (B) POEMS-flow in representative cases of POEMS syndrome are shown. In MM-flow, CD38⁺ (brightly positive)/CD138⁺/CD45^−~+ (negative to positive)/CD19⁻/CD56^± PCs are analyzed in a panel of cytoplasmic immunoglobulin (cy-Ig) light chain expression and the light chain-restricted PCs are determined to be monoclonal PCs. In POEMS-flow, CD38 is gated broadly with dim to bright and CD45 is gated narrowly with negative to dim. CD38⁺ (broadly positive)/CD138⁺/CD45⁻(narrowly negative)/CD19⁻/CD56^± PCs are further analyzed in the panel of cy-Ig light chain expression and the light chain-restricted PCs are determined to be monoclonal PCs. In the representative case of POEMS syndrome shown in this figure, monoclonal PCs are not identified by (A) MM-flow; however, (B) POEMS-flow detects monoclonal PCs (CD38⁺/CD138⁺/CD45⁻/CD19⁻/CD56⁻/λ⁺ population indicated by an arrow). (A) The number indicates the percentage of CD38⁺/CD138⁺ cells in single cells, CD45^−~+/CD38⁺/CD138⁺ cells in CD38⁺/CD138⁺ cells, CD19⁻/CD56^± cells in CD45^−~+/CD38⁺/CD138⁺ cells, and the light chain-restricted PCs in CD38⁺/CD138⁺/CD45^−~+/CD19⁻/CD56^± PCs, respectively. (B) The number indicates the percentage of CD38⁺/CD138⁺ cells in single cells, CD45⁻/CD38⁺/CD138⁺ cells in CD38⁺/CD138⁺ cells, CD19⁻/CD56^± cells in CD45⁻/CD38⁺/CD138⁺ cells, and the light chain-restricted PCs in CD38⁺/CD138⁺/CD45^−~+/CD19⁻/CD56^± PCs, respectively. The numbers in the figures represent the percentages per single cell for each cell population. The size of monoclonal PCs identified by (B) POEMS-flow is 0.22% per single cell, and the κ/λ ratio is 0.062.

Figure 2

The immunophenotype of the monoclonal plasma cells in POEMS syndrome. The immunophenotype of CD19 and CD56 in the identified clonal PCs using (A) MM-flow (n = 9) and (B) POEMS-flow (n = 18) are shown. The size of the circle in the figure indicates the number of cases with different immunophenotypes of CD19 and CD56 (CD19⁻/CD56⁺ or CD19⁻/CD56⁻ or CD19⁺/CD56⁻). (A) Five (55.6%) were CD19⁻/CD56⁺ and three (33.3%) were CD19⁻/CD56⁻ in MM-flow. (B) Five (27.8%) were CD19⁻/CD56⁺ and 12 (66.7%) were CD19⁻/CD56⁻ in POEMS-flow. (C) A comparison of CD38 mean fluorescence intensity (MFI) between monoclonal and normal plasma cells (PCs) analyzed using POEMS-flow is shown. The CD38 MFI of monoclonal PCs was significantly lower than that of normal PCs (P = 0.005), with median MFI values of 13,074.5 (range 2355–31,472) for monoclonal PCs and 20,778 (range 3674–34,672) for normal PCs.

Figure 3

Change in the size of clones detected by POEMS-flow after treatment. Six cases in which monoclonal PCs were identified at the time of diagnosis by POEMS-flow and the available post-treatment samples are evaluated. (A) Change in the size of clones detected using POEMS-flow after treatment. The size of the clones was significantly reduced after the treatment (P = 0.031) in all six patients. (B) Change in serum vascular endothelial growth factor (VEGF) levels after treatment. Serum VEGF levels were significantly decreased after treatment (P = 0.031) in all six patients. (C) Changes in the size of clones detected using POEMS-flow, serum VEGF levels, and the status of M-protein using immunofixation electrophoresis (IFE) for each patient after treatment.