Radana Neuwirtova

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.

Differential Expression of MicroRNAs in CD34+ Cells of 5q- Syndrome

BackgroundMyelodysplastic syndrome with isolated chromosome 5q deletion (5q- syndrome) is a clonal stem cell disorder characterized by ineffective hematopoiesis. MicroRNAs (miRNAs) are important regulators of hematopoiesis and their aberrant expression was detected in some clonal hematopoietic disorders. We thus analyzed miRNA expressions in bone marrow CD34+ cells of 5q- syndrome patients. Further, we studied gene expressions of miR-143, miR-145, miR-378 and miR-146a mapped within the 5q deletion.ResultsUsing microarrays we identified 21 differently expressed miRNAs in 5q- patients compared to controls. Especially, miR-34a was markedly overexpressed in 5q- patients, suggesting its role in an increased apoptosis of bone marrow progenitors. Out of four miRNAs at del(5q), only miR-378 and miR-146a showed reduced gene expression in the patients. An integrative analysis of mRNA profiles and predicted putative targets defined potential downstream targets of the deregulated miRNAs. The list of targets included several genes that play an important role in the regulation of hematopoiesis (e.g. KLF4, LEF1, SPI1).ConclusionsThe study demonstrates global overexpression of miRNAs is associated with 5q- phenotype. Identification of hematopoiesis-relevant target genes indicates that the deregulated miRNAs may be involved in the pathogenesis of 5q- syndrome by a modulation of these targets. The expression data on miRNAs at del(5q) suggest the presence of mechanisms for compensation of a gene dosage.

Myelodysplastic and myeloproliferative type of chronic myelomonocytic leukemia — distinct subgroups or two stages of the same disease?

Several authors have tried to solve the problems in the classification of CMML. A fully suitable classification does not exist. The goal of our study was to determine common and different signs of MD and MP type of CMML and to observe frequency of shifts from MD to MP-CMML. Sixty nine CMML patients were divided according to FAB proposal into two groups: 31 patients into the MD group (WBC < or = 13 x 10(9)/l) and 38 patients into the MP group (WBC < or = 13 x 10(9)/l). Presenting features and the course of the disease in both groups were evaluated. The median age of patients was not different in both groups (71.5 and 74 years, respectively), male/female ratio was 1.1 and 2.4, respectively. The median follow-up time was 15.5 months (1-58.8) in MP group and 24 months (2-118) in MD group. In MP group splenomegaly, hepatomegaly, lymphadenopathy, abnormal karyotype and skin involvement were found more often than in MD group. Median LDH value was higher in MP group. Probability of survival was higher in the MD group than in MP group (median 30 and 11 months, respectively). Leukaemia transformation frequency was similar in both groups. In 12 out of 24 (50%) MD group patients WBC increased during the course of the disease over 13 x 10(9)/l. Oscillation of WBC values below and over 13 x 10(9)/l was observed in three patients. During the follow-up time number of patients with splenomegaly and/or immature granulocytes in the PB increased. After inclusion of 12 patients who shifted from MD to MP group a new CMML group resulted characterised by longer median survival (17 months) due to a higher number of patients in an earlier stage of the disease. Failure of evolution of myeloproliferative signs and lower frequency of AL in the remaining group might be explained by an early stage of CMML, untimely deaths due to unrelated causes and/or by patients suffering of RA with monocytosis rather than of CMML. In summary, our data suggest, that evolution from MD-CMML to MP-CMML is a frequent event and that MD-CMML could be the early stage of CMML in most of cases. The WBC at diagnosis as the single criterion for subclassification of CMML does not seem to be fully justified. We propose that CMML should not be divided in MD and MP types and that monitoring of patients and search for other signs of myeloproliferation such as PB immature granulocytes, splenomegaly, lymphadenopathy, skin involvement, pleural or peritoneal effusions, spontaneous growth of CFU-GM in vitro should be taken in consideration for a better classification of CMML, which would have an impact on the therapeutic approach.

Oxidative DNA damage in bone marrow cells of patients with low-risk myelodysplastic syndrome.

Bone marrow aspirates of 19 patients with low-risk myelodysplastic syndromes (MDS) and 14 control subjects were collected in order to assess the level of oxidative DNA damage. Glycophorin A positive and negative cells separated by miniMACS magnetic cell sorting were subjected to single cell gel electrophoresis (comet assay) combined with enzymes of base excision repair (endonuclease III and formamido-pyrimidine-glycosylase) that specifically recognize oxidized nucleotides. Compared to controls, MDS patients exhibited a significant increase of oxidative damage to DNA which could contribute to genomic instability and disease progression.

Efficacy And Safety Of Administration Of Oral Iron Chelator Deferiprone In Patients With Early Myelodysplastic Syndrome

Forty-eight patients with early myelodysplastic syndrome (MDS) without excess of blasts, with average initial serum ferritin levels of 2739.5 μg/L (range 825–11287 μg/L), were treated with deferiprone (L1) in a daily dose of 40–90 mg/kg. Median duration of chelation treatment was 10.9 months (range 4–24 months). Chelation was effective (maintained or decreased iron stores) in 16 out of 22 patients (73%) with serum ferritin levels <2000 μg/L in contrast to only 12 out of 26 patients with serum ferritin levels >2000 μg/L. Combination of L1 with recombinant human erythropoietin (rHuEPO) (30–40 kU/week) resulted in effective chelation in five additional patients with serum ferritin levels >3000 μg/L. Incidence of adverse effects was comparable to that in thalassemic patients. Gastrointestinal symptoms represented the most frequent adverse effect of L1 therapy (37.5% of patients) that limited an effective escalation of the daily dose of the drug and led to discontinuation of the treatment for six patients. A decreased number of granulocytes was observed in five (13%) patients and agranulocytosis occurred in two patients (4%). Granulocyte counts were restored after cessation of L1 treatment and administration of granulocyte colony stimulating factor (G-CSF) in all but one patient. Administration of L1 in a daily dose of at least 75 mg/kg may represent an alternative approach in treatment of mild and moderate iron overload in MDS patients who cannot be treated with deferasirox (DFRA) or deferoxamine (DFO).

A comparative study of deferasirox and deferiprone in the treatment of iron overload in patients with myelodysplastic syndromes

One hundred thirteen patients with myelodysplastic syndromes (MDS) with <10% of bone marrow blasts received either deferiprone in a daily dose of 40-90 mg/kg (48 patients) or deferasirox in a daily dose of 10-40 mg/kg (65 patients). Median duration of treatment was 10,9 months for deferiprone and 13,7 months for deferasirox. A substantial reduction of iron stores evaluated as a decrease in serum ferritin of more than 50% of pretreatment level was achieved in 18 patients in deferasirox group (27.7%) but not in any patient treated with deferiprone, The incidence of adverse effects (mostly gastrointestinal symptoms) was similar after administration of both the drugs. The symptoms of deferasirox toxicity were mild and mostly transient and no drug related myelosuppresive effect was observed in contrast to deferiprone where agranulocytosis occurred in 4% of patients and the treatment had to be discontinued due to side effects in 20% of patients. The results confirmed the usefulness of deferasirox as an effective and safe iron chelator in MDS patients and indication of deferiprone as an alternative treatment only in patients with mild or moderate iron overload clearly not indicated for deferasirox.

High level of full-length cereblon mRNA in lower risk myelodysplastic syndrome with isolated 5q deletion is implicated in the efficacy of lenalidomide

Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q‐), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non‐5q‐), and 24 healthy controls] and from peripheral blood (38 patients with 5q‐, 52 non‐5q‐ patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q‐ lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real‐time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full‐length CRBN mRNA in lower risk 5q‐ patients are necessary for the efficacy of lenalidomide.

Involvement of deleted chromosome 5 in complex chromosomal aberrations in newly diagnosed myelodysplastic syndromes (MDS) is correlated with extremely adverse prognosis

MDS with complex chromosomal aberrations (CCA) are characterized by short survival and a high rate of transformation to AML. A comprehensive genome-wide analysis of bone-marrow cells of 157 adults with newly diagnosed MDS and CCA revealed a large spectrum of nonrandom genomic changes related to the advanced stages of MDS. Chromosome shattering, probably resulting from chromothripsis, was found in 47% of patients. Deleted chromosome 5 was unstable and often involved in different types of cryptic unbalanced rearrangements. No true monosomy 5 was observed. Patients with CCA involving deleted chromosome 5 had an extremely poor prognosis (median overall survival, 2 months).

Effect of erythropoietin on hepcidin expression in hemojuvelin-mutant mice

Transcription of the hepcidin (Hamp) gene is controlled by iron stores and the rate of erythropoiesis. Functional hierarchy between these two stimuli has not yet been completely established. It is also not known whether the erythropoiesis-related downregulation of Hamp expression utilises the bone morphogenetic protein/hemojuvelin (Bmp/Hjv) pathway. Hemojuvelin-mutant (Hjv-/-) mice treated with erythropoietin (EPO) at 50IU/mouse/day for three days displayed marked decrease in Hamp mRNA, demonstrating that hemojuvelin is not an indispensable component in EPO-induced Hamp gene downregulation. Irradiation of Hjv-/- mice prevented the EPO-induced decrease of Hamp mRNA, highlighting the role of erythropoiesis in Hamp gene regulation by EPO. After a single injection of EPO, Hamp mRNA levels were not significantly changed at 6h, but decreased at 10 and 24h. Chronic bleeding decreased hepatic Bmp6 mRNA levels; however, repeated EPO treatment did not change Bmp6 mRNA, suggesting that the erythropoietic regulator(s) act independently of the Bmp/Hjv pathway. Pretreatment of C57BL/6 mice with iron (5mg/mouse) almost completely inhibited the EPO-induced decrease of Hamp mRNA. This result suggests that administration of EPO to patients with transfusional iron overload is probably not associated with the risk of additional absorption of substantial amounts of iron from the diet.

DNA repair gene variants are associated with an increased risk of myelodysplastic syndromes in a Czech population

BackgroundInteractions between genetic variants and risk factors in myelodysplastic syndromes are poorly understood. In this case–control study, we analyzed 1 421 single nucleotide polymorphisms in 408 genes involved in cancer-related pathways in 198 patients and 292 controls.MethodsThe Illumina SNP Cancer Panel was used for genotyping of samples. The chi-squared, p-values, odds ratios and upper and lower limits of the 95% confidence interval were calculated for all the SNPs that passed the quality control filtering.ResultsGene-based analysis showed nine candidate single nucleotide polymorphisms significantly associated with the disease susceptibility (q-value < 0.05). Four of these polymorphisms were located in oxidative damage/DNA repair genes (LIG1, RAD52, MSH3 and GPX3), which may play important roles in the pathobiology of myelodysplastic syndromes. Two of nine candidate polymorphisms were located in transmembrane transporters (ABCB1 and SLC4A2), contributing to individual variability in drug responses and patient prognoses. Moreover, the variations in the ROS1 and STK6 genes were associated with the overall survival of patients.ConclusionsOur association study identified genetic variants in Czech population that may serve as potential markers for myelodysplastic syndromes.