Biological significance of proliferation, apoptosis, cytokines, and monocyte/macrophage cells in bone marrow biopsies of 145 patients with myelodysplastic syndrome.

Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(TGF-beta), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-alpha was correlated with TGF-beta (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-beta level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model,TGF-beta emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-alpha, TGF-beta also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.

Excessive apoptosis, increased phagocytosis, nuclear inclusion bodies and cylindrical confronting cisternae in bone marrow biopsies of myelodysplastic syndrome patients.

Transmission electron microscopic (TEM) studies have not been reported in bone marrow (BM) biopsies of patients with myelodysplastic syndromes (MDS) owing to failure to overcome the technical impediment of maintaining ultrastructural detail in decalcified tissue. Using a modified technique to physically separate pieces of bone from marrow tissue under a dissecting microscope, and embedding the material directly for TEM, ultrastructural studies were performed in 15 MDS patients and four normal BM biopsies. Biopsy tissue was also used to initiate long-term in vitro cultures and 12-week plates were sacrificed for TEM analysis. Features noted in freshly obtained decorticated tissue included an excessive apoptosis in both haematopoietic and stromal cells, ringed sideroblasts with iron-laden mitochondria and highly active, enormously increased phagocytosis. In addition, type IV nuclear inclusion body variants (NIB-v) and confronting cylindrical cisternae (CCC) were readily identified in up to 40% of stromal cells in vivo, providing an important footprint of a possible infectious agent in the pathology of MDS. Cultured stromal cells did not show excessive apoptosis and only 2-4% fibroblasts showed the presence of NIB-v or CCC, underscoring the artificial nature of ex vivo systems. We conclude that ultrastructure studies using decorticated tissue can be a powerful tool to investigate the biology and aetiology of a variety of haematopoietic disorders as it enables the direct examination of BM biopsies with their intimate stromal parenchymal cell associations preserved intact.

Increased incidence of mitochondrial cytochrome c-oxidase gene mutations in patients with myelodysplastic syndromes.

Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c-oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age-matched controls. Cytochrome c-oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential 'hot-spots'. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age-matched controls in all cell fractions (P < 0.05), with bone marrow high-density fraction (BMHDF) showing a higher mutation rate than other fractions (P < 0.05). MDS marrows showed higher levels of apoptosis than normal controls (P < 0.05), and apoptosis in BMHDF was directly related to cytochrome c-oxidase I gene mutations (P < 0.05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron-laden mitochondria. These results suggest a role for mt-DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.