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Background and Objectives: We examined the involvement of apoptosis with myelodysplastic syndrome (MDS) accompanied by peripheral cytopenias despite normo-hypercellular bone marrow., Materials and Methods: Bone marrow smears from 31 patients with MDS-refractory anemia (RA) and five normal controls were stained using the in situ end labeling (ISEL) method. Next, the inhibitory effects of a caspase-3 inhibitor, matrix metalloproteinase inhibitor (MMPI), anti-tumor necrosis factor (TNF)-alpha or anti-Fas antibody upon the apoptosis induction in overnight cultures of bone marrow cells from the patients were examined. Further, TNF-alpha, transforming growth factor (TGF)-beta and soluble Fas ligand (sFasL) concentrations in culture supernatants of the cells were assessed by enzyme-linked immunosorbent assay (ELISA)., Results: The incidence of ISEL-positive cells among MDS patients was significantly higher than in normal controls (50.8 +/- 14.0% vs. 11.3 +/- 2.4%; P < 0.0001). A caspase-3 inhibitor reduced significantly the ISEL-positive rates (32.6 +/- 15.2% vs. 50.2 +/- 16.5%; P < 0.0001). Anti-TNF-alpha or anti-Fas antibody reduced the ISEL-positive rates significantly (28.2 +/- 6.0%, 29.2 +/- 5.8%, vs. 44.2 +/- 3.4%, P < 0.001, P = 0.001, respectively). KB-R7785 also significantly decreased the ISEL-positive rates (18.0 +/- 9.3% vs. 43.6 +/- 14.0%; P < 0.0001). The concentration of TNF-alpha was significantly reduced by KB-R7785 (P < 0.05), whereas that of TGF-beta was not. Concentration of sFasL was under detectable level in the present assay system. The derivatives of KB-R7785 that can be administrated orally showed inhibitory effect on apoptosis induction as well., Conclusions: These findings suggest that MMPIs inhibits the apoptosis induction of MDS bone marrow cells via the inhibition of TNF-alpha and probably sFasL secretion, and that MMPIs can be used to control the abnormal induction of apoptosis in MDS.

CD34/QBEND10 immunostaining has been assessed in 150 bone marrow biopsies (BMB) including 91 myelodysplastic syndromes (MDS), 16 MDS-related AML, 25 reactive BMB, and 18 cases where RA could neither be established nor ruled out. All cases were reviewed and classified according to the clinical and morphological FAB criteria. The percentage of CD34-positive (CD34 +) hematopoietic cells and the number of clusters of CD34+ cells in 10 HPF were determined. In most cases the CD34+ cell count was similar to the blast percentage determined morphologically. In RA, however, not only typical blasts but also less immature hemopoietic cells lying morphologically between blasts and promyelocytes were stained with CD34. The CD34+ cell count and cluster values were significantly higher in RA than in BMB with reactive changes (p<0.0001 for both), in RAEB than in RA (p=0.0006 and p=0.0189, respectively), in RAEBt than in RAEB (p=0.0001 and p=0.0038), and in MDS-AML than in RAEBt (p<0.0001 and p=0.0007). Presence of CD34+ cell clusters in RA correlated with increased risk of progression of the disease. We conclude that CD34 immunostaining in BMB is a useful tool for distinguishing RA from other anemias, assessing blast percentage in MDS cases, classifying them according to FAB, and following their evolution.

Nucleoli of erythroblasts have been studied in patients suffering from refractory anemia (RA) of myelodysplastic syndrome (MDS) and in control patients without a disturbed erythropoiesis in order to provide information on the incidence of nucleoli and micronucleoli in these cells. Nucleoli in erythroblasts were visualized by a simple cytochemical procedure for the demonstration of RNA which facilitated the visualization not only large nucleoli but also micronucleoli in advanced stages of the erythroblastic maturation. In control patients nucleoli were detected in all stages of erythroblastic development. In patients suffering from RA of MDS, a relatively large population of polychromatic and orthochromatic erythroblasts was characterized by a loss of nucleoli accompanied by the decreased incidence of micronucleoli characteristic of these cells. In contrast to control patients, in patients suffering from RA of MDS the number of nucleoli expressed by the values of the nucleolar coefficient of erythroblasts was smaller, particularly in both the early and terminal stages of erythroblastic development. Thus in patients with RA of MDS both the abnormal loss of nucleoli and decreased number of nucleoli in erythroblasts apparently represent and reflect a further abnormality of disturbed erythropoiesis.

IFN alpha alone or in combination with retinoids or haematopoietic growth factors has been used to treat patients with early MDS because of its properties as a differentiation inducing agent. We investigated whether treatment of patients with refractory anemia (RA) with IFN alpha (1.5x10(6) IU twice a week) and intermittent all-trans retinoic acid (ATRA, 25 mg/m2/d) influences in-vitro megakaryocytic (MK) proliferation and differentiation stimulated by PEG-rHuMGDF. Low-density non-adherent bone marrow (BM) cells from 8 patients with RA were assayed prior to any treatment other than supportive and after a period of 6 months of treatment. MK development was assayed in suspension cultures in the presence of PEG-rHuMGDF and SCF for 7 d using morphological criteria and flow cytometric analysis of CD42b (GP1b) positive cells. BM-cells from 10 healthy individuals served as control. Following stimulation with PEG-rHuMGDF 23+/-7% and 16+/-4% of control cells were CD42b positive after 5 and 7 d of cultures, respectively. In cultures of cells from MDS patients prior to treatment 8+/-2% and 7+/-3% of cells were CD42b+ on days 5 and 7. In the course of IFN alpha treatment cultures of all BM samples from these MDS patients revealed a significant reduction of MK precursor cells (3+/-2%, CD42b+, p=0.03 and 0.04). In conclusion, treatment with TFN alpha and ATRA did not result in improved megakaryocytopoiesis as assessed by in-vitro cultures. On the contrary, low-dose IFN alpha appears to suppress cell proliferation as well as MK development.

In order to evaluate the diagnostic importance of the megakaryocytic morphology in the 5q- syndrome we studied the bone marrow from 48 unselected patients with myelodysplastic syndromes (MDS). 44 cases were primary and 4 secondary to cytostatic drug treatment or irradiation. There were 24 cases with chromosome anomalies, of whom 10 had del (5q). 4 of these had refractory anaemia (RA) with 5q- as the sole anomaly (group A), 2 had RA with 5q- and additional chromosome anomalies consisting of trisomy 8 (group B); 3 patients had RA with excess of blasts (RAEB) and complex, karyotypic changes also including 5q- (group B). Changes of the same type were found in 1 case of multiple myeloma with secondary MDS. All 6 RA patients with 5q- had characteristic megakaryocytes. More than 50% of the cells had no more than 2 nuclear segments, and predominantly had a diameter of 30 micron or more. No other patient with RA showed this picture. Only 1 patient with RAEB 5q- in group B had the same megakaryocytic changes. We conclude that diagnosis of a 5q- syndrome may be strongly suspected in cases of RA with these bone marrow changes. In cases of RAEB 5q- group B the bone marrow examination did not reveal the same consistent changes.

34 patients with primary myelodysplastic syndrome (MDS), initially diagnosed as subtypes refractory anaemia (RA) and RA with ringed sideroblasts (RA-S), were followed to investigate the distribution of lymphoid and myeloid differentiation antigens in the blood and bone marrow in search of potential prognostic significance with regard to progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia, and for relations to clinical, morphological and cytogenetic findings. Patients who later progressed to RAEB had significantly decreased percentages of anti-T8 positive T-suppressor cells in the blood at diagnosis compared to those who did not (p = 0.05). Sequential analysis showed a decrease with time also in the percentages of anti-T8-positive cells (p = 0.05). In the bone marrow, progressing patients initially showed significantly increased percentages of anti-My9-positive immature myeloid cells (p less than 0.001), and the percentages of anti-My9-positive cells in the bone marrow increased with time (p less than 0.005). Analysis of the pooled data revealed a statistically significant relation between increasing percentages of anti-My9-positive cells and the frequencies of clonally abnormal (p less than 0.001) and abnormal (p = 0.004) metaphases.

In vitro megakaryocytic colony formation by progenitors from the bone marrow was studied in 40 myelodysplastic syndrome patients. Megakaryocytic colonies were decreased in number or absent in 30 patients; only 10 showed normal colony growth. The growth correlated to some extent with the FAB-class. Patients with normal colony formation had either RA or RARS, but also in these two FAB-types half of the patients showed reduced megakaryocytic colony formation. Only 1 out of 15 patients with RAEB or RAEBt had normal megakaryocyte growth. The patients with CMML did not show any megakaryocytic colonies. The growth of erythroid colonies was normal in 3 patients and reduced or absent in the others. All 3 with normal erythroid colony formation also showed normal megakaryocyte growth, and all patients with normal megakaryocyte colony formation also had normal granulocyte-macrophage colony growth. Granulocyte-macrophage colony and cluster formation was normal in 17 patients. Defective formation of megakaryocytic, erythroid, and granulocyte-macrophage colonies was seen in 22 patients, compatible with a defect in a pluripotent stem cell. Megakaryocytic colony formation had no obvious correlation with any specific chromosome abnormality, and the distribution of the growth patterns was almost similar in patients with a normal and those with an abnormal karyotype.