Characterization of defective megakaryocytic development in patients with myelodysplastic syndromes

Megakaryocytic differentiation of progenitor cells was investigated in nine patients with low-risk myelodysplastic syndromes (MDS) (eight refractory anemia [RA] and one RA with ringed sideroblasts [RARS]) and five patients,vith high-risk MDS (two RA with excess of blasts [RAEB] and three RAEB in transformation [RAEB-T]), Bone marrow-derived CD34(+) cells were enriched to a purity of 87% +/- 2% (mean +/- SEM) and assayed in short-term suspension cultures in the presence of 10 ng/mL of PEGylated recombinant human megakaryocyte (MK) growth and development factor (PEG-rHuMGDF) and in addition to 50 ng/mL stem cell factor and 10 ng/mL interleukin-3, Cells of the megakaryocytic lineage were identified by flow cytometric analysis of CD42b (GP1b) and mature MKs by morphologic criteria. Transcription of c-mpl receptor-specific mRNA in the CD34(+) cells of these patients was investigated by full-length reverse transcriptase polymerase chain reaction of the p form of c-mpl as well as of the alternative splice product c-mpl k, CD34(+) cells from seven healthy bone marrow donors served as controls. Differentiation along the MK pathway was stimulated in five patients with RA, C-mpl mRNA was expressed in the CD34(+) cells in all cases, In three low-risk patients the capacity for in vitro MK growth was absent or minimal even though mRNA for c-mpl receptor was detected in the CD34(+) cells of this group as well, In patients with high-risk MDS, PEG-rHuMGDF stimulated in vitro MK growth from CD34(+) cells in only one of five cases. As in the patients with low-risk MDS, c-mpl mRNA for both c-mpl p and c-mpl k splicing products was detected. These results indicate that the in vitro response to stimulation with c-mpl ligand discriminates between two groups of patients with low-risk MDS and that the observed defect in megakaryocytic development is unrelated to the level of c-mpl expression in both low-risk and high-risk MDS. (C) 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.