To assess biologic characteristics of marrow progenitors in MDS, we studied a consecutive series of MDS patients receiving no current treatment.

Methods: Patients’ (n=17) clinical histories, lab data, cytogenetics, and morphology were reviewed for confirmation of diagnosis and blinded to study results. Patients lacking primary marrow disease (n=9) were used as controls. Marrow samples were studied at 2-5 hours after procurement (mean 3.1 hours), and re-assessed at 24 hours. A small number of marrow samples were fixed within minutes of procurement to validate findings. Each sample was assessed for CD45 antigen density, log side scatter, CD34, CD71, DRAQ 5 (DNA content), MPM-2, p-H2A.X (serine 139), and Annexin V with an FC500 (Beckman-Coulter) flow cytometer. Analysis was performed using Winlist 5.0 software (Verity Software) with DDE links to ModfitLT3.0 using modifications of published methods along with Esoterix Center for Innovation generated algorithms. Analyses were blinded to clinical results. Results: Most MDS patients demonstrated increased S-phase cell cycle fractions. Despite this, neither myeloid nor erythroid progenitors in MDS demonstrated an increased mean mitotic index vs. normals (0.315/0.359 and 1.33/1.48 respectively), and 7 MDS patients demonstrated no mitotic events in myeloid precursors (never observed in the normal dataset). DNA damage (H2A.X binding) was increased in myeloid and erythroid progenitors in MDS vs. normals (19/4% and 9/1.6% respectively). Annexin V staining was modestly elevated in maturing progenitors in MDS patients vs. normals (16.4% vs. 10.9%), and increased 4% in each group at 24 hours. Stem cells (CD34+) and lymphocytes were negative for both H2A.X and Annexin V binding. Discussion: These results indicate abnormalities in MDS marrow biology that may be useful as diagnostic tests for MDS. They suggest a model of MDS characterized by DNA damage in maturing hematopoietic progenitors in MDS, with late cell cycle arrest, accumulation of cells in S or G2, and decreased cell division (mitotic events) despite cellular marrow. In this model, DNA damage may initiate apoptosis in cells, but the findings are inconsistent with massive successful programmed cell death as a central event in MDS pathogenesis. The model has therapeutic implications for MDS.

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