Accumulation of homoplasmic mtDNA point mutations in erythroblasts isolated from the bone marrow of patients with refractory anemia with ring sideroblasts (RARS).

It was hypothesised that mitochondrial iron overload in patients with refractory anemia with ring sideroblasts (RARS) results from mitochondrial DNA (mtDNA) mutations. To analyse the mtDNA sequence of iron storing mitochondria sensitively, we developed new protocols for selective erythroblasts isolation, mtDNA PCR amplification and sequencing. Using this approach, we found in each of the three RARS patients examined a unique spectrum of homoplasmic mtDNA point mutations affecting several mtDNA genes. Prediction analyses suggest that identified mutations do not result in major perturbations of mitochondrial functions and are tolerated. We discuss a mechanism explaining how the mutations identified may contribute to RARS pathogenesis.

[Rearrangements of 11q in patients with hematologic malignancies]. / Prestavby 11q u nemocných s hematologickými malignitami.

BACKGROUND: The aim of this study was to investigate the rearrangement of MLL gene in bone marrow cells of patients with hematological malignancies with various types of 11q aberrations. These aberrations have been observed in acute lymphoblastic and acute myeloid leukemias as well as in myelodysplasias and lymphomas. METHODS AND RESULTS: Correlations of clinical characteristics, type of aberrations, diagnoses and survival of patients were evaluated. Using classical cytogenetic techniques we found 11q aberration in 17 patients with different hematological malignancies. FISH with dual color locus specific probe for MLL gene was used to confirm or exclude the rearrangement of this gene. Whole chromosome painting probes and multicolor FISH were performed for identification of chromosomes involved in complex translocations. Balanced rearrangements of 11q were found in 3 patients, in 14 patients unbalanced aberrations were found with rearrangement (4), deletion (4) and amplification (2) of MLL gene. In 7 patients no rearrangement of MLL gene was found. CONCLUSIONS: Correlation between clinical characteristics, type of aberrations, diagnoses and survival of patients was not significant, therefore further studies of larger cohort of patients are necessary to evaluate the prognostic value of 11q rearrangement.

[The International Prognostic Scoring System for primary myelodysplastic syndrome]. / Mezinárodní prognostický bodovací systém u pacientu s primárním myelodysplastickým syndromem.

A number of prognostic scoring systems for patients with myelodysplastic syndrome (MDS) have been introduced since FAB classification of the MDS in 1982. Recently, the International Prognostic Scoring System (IPSS), published in 1997 by Greenberg et al. [9] is based on the percentage of bone marrow (BM) blasts, cytogenetic abnormalities and number of cytopenias. We applied criteria of the IPSS on 205 patients (pts) with primary MDS (RA = 82, RARS = 49, RAEB = 42, RAEB-t = 8, CMML = 24 pts). IPSS discriminated within each of the FAB-subgroups: RA pts were present in low risk and intermediate (Int) I and II risk subgroups, RARS pts were separated into low and Int I, RAEB were distributed predominantly between Int I and Int II risk groups, RAEB-t in high-risk group, and CMML pts were distributed in all groups. In contrary to Greenberg's group of the MDS patients there are only three risk-groups in our study: low risk (score 0-0.5), intermediate (1-2) and high risk (> 2); the median survival and the risk of the evolution to the acute leukemia (p = 0.0001) are significantly different.

[The comet assay: principles of the method and clinical utilization]. / Kometový test: princip metody a klinické aplikace.

BACKGROUND: Single cell gel electrophoresis or comet assay is used at present in the world to study of DNA damage, DNA repair and apoptosis. The aim of the work is to introduce the principle of comet assay to the medical community and to give a basic survey of its possible clinical applications. The article includes our first experience with this method in detection of apoptosis in bone marrow cells of patients with myelodysplastic syndrome (MDS). METHODS AND RESULTS: The whole bone marrow aspirates from 6 patients with MDS and 7 control persons were processed by alkaline version of comet assay and the degree of DNA fragmentation in individual cells was quantified using Image Analysis System. In comparison with controls, the patients with diagnosis RA and RARS exhibited in bone marrow significantly elevated number of cells with high level of DNA breaks, reflecting most probably the apoptotic cleavage of DNA. In contrast, the patient in proliferative stage of the disease (MDS-CMML-->AML) exhibited decreased frequency of apoptotic cells, well below the control level. CONCLUSION: Our results correspond with the data published on the occurrence of apoptosis in particular types of MDS. Comet assay represents a simple and cheap technique, applicable in clinical hematology to specify the diagnosis, to monitor the disease progress and efficacy of therapy not only in patients with MDS but also in other diseases resulting from an imbalance between proliferation and apoptosis.

Cyclosporin A therapy in hypoplastic MDS patients and certain refractory anaemias without hypoplastic bone marrow.

We report 17 cytopenic patients with myelodysplastic syndrome (MDS) of refractory anaemia (RA) subtype with hyper-, normo- or hypo-cellular bone marrow (BM), who were treated with cyclosporin A (CyA). Substantial haematological response was observed in 14 patients (82%): their anaemia improved and all transfusion-dependent patients achieved transfusion independence. Complete trilineage recovery was observed in four patients (23%). The CyA therapy has not yet failed in any of the 14 successfully treated patients during follow-up times ranging from 5 to 30 months. CyA was well tolerated in 14 patients; serious side-effects required termination of the therapy in three patients in whom the blood count rapidly deteriorated to former levels upon cessation of therapy. Two patients benefited from a combination therapy of CyA and erythropoietin. Six patients experienced various autoimmune phenomena. CyA could thus offer an alternative treatment for certain MDS patients with RA regardless of hyper-, normo- or hypo-cellularity of bone marrow (BM). The mechanism of the beneficial effect of CyA is discussed and remains the subject of an ongoing study.

[Administration of granulocyte-macrophage colony growth factors in the treatment of postcytostatic myelosuppresion in oncohematologic patients]. / Pouzití rustového faktoru pro granulocyto-makrofágovou radu v lécbe postcytostatické myelosuprese u onkohematologických nemocných.

BACKGROUND: Growth factors for granulocytic and granulocyte-macrophage series (G-CSF and GM-CSF) in the form of recombinant proteins are used in various leukopenias. The aim of this work was to follow the effect of GM-CSF (Leucomax Sandoz) in myelosuppression after the therapy with cytotoxic drugs. METHOD AND RESULTS: Leucomax was given to 22 patients with oncohematological disease (3 patients with acute lymphoblastic leukemia, 5 patients with various myeloid malignacies, 4 myelomas and 10 patients with non-Hodgkin lymphomas). In comparison to the literary data lower doses of Leucomax were used, it is 150-200 micrograms a day and patient. The length of administration of the growth factor was reduced to the shortest possible time according to the leukocyte count monitoring. Leucomax administration was started when leukocytes dropped below 1.10(9), in patients with severe leukopenias after repeated chemotherapy Leucomax was applied earlier. From 17 patients treated for sufficiently long period of time 70% responded to the GM-CSF application without any serious side-effects. In most cases, with the exception of myeloma, the growth factor was applied for 4-10 days. The leukocyte increase above 3 x 10(9) appeared in 3-8 days after the start of the treatment. CONCLUSIONS: Correctly indicated application of growth factors in postcytostatic myelosuppression is a great contribution to antitumor therapy. According to our experience in most cases 5-7 days of the daily dose of 150-200 micrograms of GM-CSF is sufficient for the stimulation of leukopoiesis. In postherapeutic myelosuppression in acute myeloblastic leukemia the growth factor should be used only as a life saving therapy since it can stimulate the leukemic cell population.