Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndrome associated with chromosome 5q deletion

Abstract

Background: Lenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodysplastic syndrome and interstitial deletion of the long arm of chromosome 5 [del(5q)]. The aim of this study was to explore the effect of lenalidomide treatment on the reserves and functional characteristics of bone marrow hematopoietic progenitor/precursor cells, bone marrow stromal cells and peripheral blood lymphocytes in patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q).

Design and methods: We evaluated the number and clonogenic potential of bone marrow erythroid/myeloid/megakaryocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesion molecule expression of CD34(+) cells by flow cytometry, the hematopoiesis-supporting capacity of bone marrow stromal cells using long-term bone marrow cultures and the number and activation status of peripheral blood lymphocytes in ten patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q) receiving lenalidomide.

Results: Compared to baseline, lenalidomide treatment significantly decreased the proportion of bone marrow CD34+ cells, increased the proportion of CD36(+)/GlycoA(+) and CD36(-)/GlycoA(+) erythroid cells and the percentage of apoptotic cells within these cell compartments. Treatment significantly improved the clonogenic potential of bone marrow erythroid, myeloid, megakaryocytic colony-forming cells and increased the proportion of CD34(+) cells expressing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48. The hematopoiesis-supporting capacity of bone marrow stroma improved significantly following treatment, as demonstrated by the number of colony-forming cells and the level of stromal-derived factor-1 alpha and intercellular adhesion molecule-1 in long-term bone marrow culture supernatants. Lenalidomide treatment also increased the proportion of activated peripheral blood T lymphocytes.

Conclusions: The beneficial effect of lenalidomide in patients with lower risk myelodysplastic syndrome with del(5q) is associated with significant increases in the proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant alterations in the adhesion profile of bone marrow CD34(+) cells.

Figure 1.

Flow-cytometric analysis of BM cells. (A) Scattergram of forward scatter (FSC) versus side scatter (SSC) to allow gating on cells with low FSC and low SSC properties (R1). (B) Scattergram of anti-CD34 fluorescence versus SSC gated on R1, to allow gating on CD34⁺ cells (R2). (C) Scattergram of FSC versus 7AAD fluorescence gated on the CD34⁺ cells (R2) showing the live (R3), early apoptotic (R4) and late apoptotic/dead (R5) cells. Apoptosis was similarly studied within the CD34⁺/CD71⁺ erythroid progenitor cells (R6) (plot D) and the CD36⁺/GlycoA⁺ (R7) and CD36⁻/GlycoA⁺ (R8) erythroid precursor cells (plot E). An example of apoptosis in the ungated cells is depicted in scattergram (F) to show the live (R9), early (R10) and late apoptotic/dead (R11) cells.

Figure 2.

Flow-cytometric evaluation of the reserves and survival characteristics of BM progenitor and erythroid precursor cells before and after lenalidomide therapy. The left bars represent the mean proportion (± SEM) of BM CD34⁺ progenitor and CD36⁺/GlycoA⁺ and CD36⁻/GlycoA⁺ erythroid precursor cells before and after treatment. The right bars represent the mean proportion (± SEM) of apoptotic (7AAD^dim) cells within the CD34⁺, CD36⁺/GlycoA⁺ and CD36⁻/GlycoA⁺ cell compartments before and after therapy. Results were compared using the Student’s t test for paired samples and P values are indicated. SEM: standard error of the mean.

Figure 3.

BM clonogenic progenitor cells and peripheral blood lymphocyte subsets before and after lenalidomide therapy. (A) The bars in the upper graph represent the mean number (±SEM) of CFU-Meg, CFU-GM and BFU-E colonies obtained from 10⁷ BMMC. The bars in the lower graph represent the mean CFC numbers (±SEM) in LTBMC supernatants over 5 weeks of culture, before and after therapy. Results were compared using Student’s t test for paired samples (upper graph) and 2-way ANOVA (lower graph) and the P and F values are indicated. (B) The bars in graph B represent the mean proportion (±SEM) of PB lymphocyte subpopulations (upper panel) and the mean percentage of cells expressing markers of activation within the CD3⁺ cell fraction (lower panel), before and after therapy. Results were compared using Student’s t test for paired samples and P values are shown. SEM; standard error of the mean.