Lenalidomide treatment in lower risk myelodysplastic syndromes-The experience of a Czech hematology center. (Positive effect of erythropoietin ± prednisone addition to lenalidomide in refractory or relapsed patients).

Lenalidomide therapy represents meaningful progress in the treatment of anemic patients with myelodysplastic syndromes with del(5q). We present our initial lenalidomide experience and the positive effect of combining erythropoietin and steroids with lenalidomide in refractory and relapsed patients. We treated by lenalidomide 55 (42 female; 13 male; median age 69) chronically transfused lower risk MDS patients with del(5q) (45) and non-del(5q) (10). Response, meaning transfusion independence (TI) lasting ≥ eight weeks, was achieved in 38 (90%) of analyzed patients with del(5q), of whom three achieved TI only by adding erythropoietin ±â€¯prednisone. Another five patients responded well to this combination when their anemia relapsed later during the treatment. In the non-del(5q) group only one patient with RARS-T reached TI. Cytogenetic response was reached in 64% (32% complete, 32% partial response). The TP53 mutation was detected in 7 (18%) patients; four patients progressed to higher grade MDS or acute myeloid leukemia (AML). All seven RAEB-1 patients cleared bone marrow blasts during lenalidomide treatment and reached complete remission (CR); however, three later progressed to higher grade MDS or AML. Lenalidomide represents effective treatment for del(5q) group and combination with prednisone and erythropoietin may be used for non-responders or therapy failures.

TP53 mutation variant allele frequency is a potential predictor for clinical outcome of patients with lower-risk myelodysplastic syndromes.

TP53 mutations are frequently detected in patients with higher-risk myelodysplastic syndromes (MDS); however, the clinical impact of these mutations on the disease course of patients with lower-risk MDS is unclear. In this study of 154 lower-risk MDS patients, TP53 mutations were identified in 13% of patients, with prevalence in patients with del(5q) (23.6%) compared to non-del(5q) (3.8%). Two-thirds of the mutations were detected at the time of diagnosis, and one-third were detected during the course of the disease. Multivariate analysis demonstrated that a TP53 mutation was the strongest independent prognostic factor for overall survival (OS) (HR: 4.39) and progression-free survival (PFS) (HR: 3.74). Evaluation of OS determined a TP53 variant allele frequency (VAF) threshold of 6% as an optimal cut-off for patient stratification. The median OS was 43.5 months in patients with mutations detected at the time of diagnosis and a mutational burden of > 6% VAF compared to 138 months (HR 12.2; p = 0.003) in patients without mutations; similarly, the median PFS was 20.2 months versus 116.6 months (HR 79.5; p < 0.0001). In contrast, patients with a mutational burden of < 6% VAF were stable for long periods without progression and had no significant impact on PFS or OS. Additionally, we found a high correlation in the mutational data from cells of the peripheral blood and those of the bone marrow, indicating that peripheral blood is a reliable source for mutation monitoring. Our results indicate that the clinical impact of TP53 mutations in lower-risk MDS patients depends on the level of mutational burden.

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).

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.

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

BACKGROUND: Interactions 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. METHODS: The 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. RESULTS: Gene-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. CONCLUSIONS: Our association study identified genetic variants in Czech population that may serve as potential markers for myelodysplastic syndromes.

Transcription factors Fli1 and EKLF in the differentiation of megakaryocytic and erythroid progenitor in 5q- syndrome and in Diamond-Blackfan anemia.

Friend leukemia virus integration 1 (Fli1) and erythroid Krüppel-like factor (EKLF) participate under experimental conditions in the differentiation of megakaryocytic and erythroid progenitor in cooperation with other transcription factors, cytokines, cytokine receptors, and microRNAs. Defective erythropoiesis with refractory anemia and effective megakaryopoiesis with normal or increased platelet count is typical for 5q- syndrome. We decided to evaluate the roles of EKLF and Fli1 in the pathogenesis of this syndrome and of another ribosomopathy, Diamond-Blackfan anemia (DBA). Fli1 and EKLF mRNA levels were examined in mononuclear blood and bone marrow cells from patients with 5q- syndrome, low-risk MDS patients with normal chromosome 5, DBA patients, and healthy controls. In 5q- syndrome, high Fli1 mRNA levels in the blood and bone marrow mononuclear cells were found. In DBA, Fli1 expression did not differ from the controls. EKLF mRNA level was significantly decreased in the blood and bone marrow of 5q- syndrome and in all DBA patients. We propose that the elevated Fli1 in 5q- syndrome protects megakaryocytic cells from ribosomal stress contrary to erythroid cells and contributes to effective though dysplastic megakaryopoiesis.

Changes associated with lenalidomide treatment in the gene expression profiles of patients with del(5q).

UNLABELLED: We used microarray profiling to investigate the direct effects of lenalidomide on gene expression in isolated CD14(+) monocytes from 6 patients with del(5q). Our data demonstrate that changes in genes involved the tumor necrosis factor (TNF) signaling pathway and the bone marrow stroma, suggesting that treatment with lenalidomide may help restore the damaged niche and suppress the TNF signaling pathway. BACKGROUND: Lenalidomide is an effective treatment for patients with del(5q) and myelodysplastic syndrome (MDS) The exact mechanism of lenalidomide function and its impact on the prognosis of patients is not known exactly. MATERIALS AND METHODS: We used gene expression profiling to study the effect of lenalidomide therapy in peripheral blood CD14(+) monocytes of 6 patients with del(5q) and MDS. RESULTS: After lenalidomide treatment, genes involved in the tumor necrosis factor (TNF) signaling pathway that were upregulated in the patients before treatment decreased to the healthy control baseline expression level. This change in gene expression, in conjunction with increased expression of repressed genes that affect the stem cell niche (ie, CXCR4 and CRTAP), may exert a positive effect on treated patients. In contrast, we found that increased expression of the ARPC1B gene may have a negative impact on the stability of patient remission. CONCLUSION: The observed changes in gene expression described here may contribute to the identification of pathways that are affected by lenalidomide, which may help to explain the effects of this drug.

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).

Differential expression of microRNAs in CD34+ cells of 5q- syndrome.

BACKGROUND: Myelodysplastic 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. RESULTS: Using 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). CONCLUSIONS: The 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.

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.

Nature of frequent deletions in CEBPA.

C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to the family of leucine zipper transcription factors and is necessary for transcriptional control of granulocyte, adipocyte and hepatocyte differentiation, glucose metabolism and lung development. C/EBPalpha is encoded by an intronless gene. CEBPA mutations cause a myeloid differentiation block and were detected in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma and non-Hodgkin's lymphoma (NHL) patients. In this study we identified in 41 individuals from 824 screened individuals (290 AML patients, 382 MDS patients, 56 NHL patients and 96 healthy individuals) a single class of 23 deletions in CEBPA gene which involved a direct repeat of at least 2 bp. These mutations are characterised by the loss of one of two same repeats at the ends of deleted sequence. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493-498_865-870), GCCAAGCAGC (508-517_907-916) and GG (486-487_885-886), all according to GenBank accession no. NM_004364.2. A mechanism for deletion formation between two repetitive sequences can be recombination events in the repair process. Double-stranded cut in DNA can initiate these recombination events of adjacent DNA sequences.

Defective cytotoxicity of T lymphocytes in myelodysplastic syndrome.

OBJECTIVE: The capacity of mononuclear blood cells to form autoreactive cytotoxic T lymphocytes was investigated in order to elucidate the mechanism of successful immunosuppressive therapy in some myelodysplastic syndrome (MDS) patients (autoreactivity studies). The failure in autoreactivity studies raised the question of alloreactive cytotoxic T lymphocyte formation in MDS (alloreactivity studies). MATERIALS AND METHODS: Sixteen MDS patients and relevant controls were examined. Autoreactive lymphocytes directed against autologous bone marrow mononuclear cells and alloreactive lymphocytes directed against unrelated third-party cells were tested using cytotoxicity assay. In addition, we used one-way mixed lymphocyte reaction, human androgen receptor test for clonality detection, and enzyme-linked immunosorbent assay kits for tumor necrosis factor and interferon-gamma testing. RESULTS: We did not confirm the presence of autoreactive T cells in eight of nine MDS patients tested. The response to allogeneic cells was impaired in 11 of 16 MDS patients, more often in refractory anemia (RA; 80%) than in RA with ring sideroblasts (40%). Interestingly, the response to allogeneic cells in mixed lymphocyte reaction was normal in all MDS patients. T lymphocytes were polyclonal in all but one patient. Tumor necrosis factor and interferon-gamma level in supernatants of mononuclear cells was significantly reduced in RA. CONCLUSION: The presumed autoaggressive T cells were not confirmed in MDS in our experimental arrangement. Alloreactivity studies demonstrated the impairment of effector cytotoxic phase of cell-mediated immunological reaction in MDS, namely in RA. The significance of our finding of defective cytotoxicity for pathogenesis, clinical course, and even for therapy is discussed together with other immunological defects reported so far.

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.

DNA instability in low-risk myelodysplastic syndromes: refractory anemia with or without ring sideroblasts.

We tested genomic instability in patients with myelodysplastic syndrome (MDS) by the comet assay and verified the suitability of this approach as a tool for analysis of ineffective hematopoiesis in refractory anemia (RA) and RA with ring sideroblasts (RARS). Erythroid and myeloid cell populations from bone marrow aspirates of 20 RA, 14 RARS and 15 control subjects were separated by differential expression of glycophorin A and subjected to comet assay. The extent of DNA migration was measured in single cells (200 cells/bone marrow fraction/subject). The results were in agreement with the concept of increased apoptosis in low-risk MDS subtypes. The RA samples had a significantly higher DNA instability than controls in glycophorin A positive cells, and the extent of DNA breakage correlated with the degree of cytopenia. Although RARS had an even higher rate of genomic instability in bone marrow cells than RA, there was no clear relationship to peripheral cytopenia. This suggests an additional DNA instability of non-apoptotic origin. Whether this increase is associated with an increased repair of oxidative damage in DNA arising due to iron deposits in ring sideroblasts remains to be formally proven. Comet assay provides a promising tool for the investigation of difference between RA and RARS pathobiology.

CEBPA polymorphisms and mutations in patients with acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and non-Hodgkin's lymphoma.

The transcription factor CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor which is required for normal myeloid differentiation. C/EBPalpha is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. C/EBPalpha is a member of the basic region leucine zipper (bZIP) class of DNA-binding proteins. The loss of function of C/EBPalpha has leukemogenic potential. Four types of polymorphisms and 25 mutations (3 already known mutations and 22 novel mutations) were detected in CEBPA (gene for the transcription factor CCAAT/enhancer binding protein (C/EBP) alpha) in analysed samples from 390 patients with myelodysplastic syndrome (MDS) and hematologic malignancies. CEBPA mutations were found in 14/152 (9.2%) of acute myeloid leukemia (AML) patients' samples, 6/143 (4.2%) of MDS patients' samples, 2/56 (3.6%) of non-Hodgkin's lymphoma (NHL) patients' samples and 2/39 (5.1%) of multiple myeloma (MM) patients' samples. No C/EBPalpha mutations were detected in healthy donors (41 individuals). We discuss how these mutations can affect the cellular function of C/EBPalpha and block the myeloid differentiation.

Prognostic significance of del(20q) in patients with hematological malignancies.

Deletions of the long arm of chromosome 20 represent a common chromosomal abnormality associated with myeloid malignancies, in particular with myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). Using G-banding cytogenetic techniques, we found clones with del(20q) in 36 patients with hematological malignancies examined in our laboratory during the years 2001-2003: in 23 patients as a sole cytogenetic aberration and in 13 patients together with other chromosomal changes. Fluorescence in situ hybridization (FISH) with a probe specific for the 20q12 region was used in all cases to confirm the presence of the clone with deletion. For patients with additional or complex chromosomal rearrangements, multicolor FISH (M-FISH) analysis was performed. Statistical evaluation of the prognostic impact of sex, age, diagnosis, and karyotype was performed. The survival time correlated with the type of chromosomal aberration; no significant differences in survival were found for sex, age, and diagnosis.