Florian Nolte - Academia.edu (original) (raw)

Papers by Florian Nolte

Research paper thumbnail of A molecular risk score integrating BAALC, ERG and WT1 expression levels for risk stratification in acute promyelocytic leukemia

Leukemia Research, 2015

To date risk stratification in acute promyelocytic leukemia (APL) is based on highly dynamic leuk... more To date risk stratification in acute promyelocytic leukemia (APL) is based on highly dynamic leukocyte and platelet counts only. To identify a more robust risk stratification model, a molecular risk score was developed based on expression levels of the genes BAALC, ERG and WT1. Hereby, the main focus was on prediction of relapse. The integrative risk score divided patients into two groups with highly significant differences in outcome. It discriminated a high risk group with a high incidence of relapse successfully from a low risk group with no APL-related events after achievement of first remission. Especially the concurrent presence of molecular risk factors showed to be a negative prognostic factor in APL. The molecular risk score might be a promising approach to guide monitoring of APL patients and therapeutic decisions in the future.

Research paper thumbnail of Telomere dynamics in patients with del (5q) MDS before and under treatment with lenalidomide

Leukemia Research, 2015

Myelodysplastic syndrome (MDS) associated with an acquired, isolated deletion of chromosome 5q (d... more Myelodysplastic syndrome (MDS) associated with an acquired, isolated deletion of chromosome 5q (del (5q) MDS), represent a clonal disorder of hematopoiesis and a clinically distinct entity of MDS. Treatment of del (5q) MDS with the drug lenalidomide has significantly improved quality of life leading to transfusion independence and complete cytogenetic response rates (CCR) in the majority of patients. Telomeres are located at the end of eukaryotic chromosomes and are linked to replicative history/potential as well as genetic (in) stability of hematopoietic stem cells. Here, we analyzed telomere length (TL) dynamics before and under lenalidomide treatment in the peripheral blood and/or bone marrow of del (5q) patients enrolled in the LEMON-5 study (NCT01081431). Hematopoietic cells from del (5q) MDS patients were characterized by significantly shortened TL compared to age-matched healthy controls. Telomere loss was more accelerated in patients with longer disease duration (>2 years) and more pronounced cytopenias. Sequential analysis under lenalidomide treatment revealed that previously shortened TL in peripheral blood cells was significantly "elongated" towards normal levels within the first six months suggesting a shift from clonal del (5q) cells towards normal hematopoiesis in lenalidomide treated MDS patients. Taken together our findings suggest that the development of the del (5q) clone is associated with accelerated telomere shortening at diagnosis. However, upon induction of CCR and reoccurrence of normal hematopoiesis, the lack of a persistent TL deficit argues against telomere-mediated genetic instability neither as a disease-promoting event of del (5q) MDS nor for lenalidomide mediated development of secondary primary malignancies of the hematopoietic system in responding patients.

Research paper thumbnail of Validation of cytogenetic risk groups according to International Prognostic Scoring Systems by peripheral blood CD34+FISH: results from a German diagnostic study in comparison with an international control group

Haematologica, 2015

International Prognostic Scoring Systems are used to determine the individual risk profile of mye... more International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%-20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34(+)) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization anal...

Research paper thumbnail of Molecular Changes in Myelodysplastic Syndrome

The Myelodysplastic Syndromes, 2011

Myelodysplastic syndromes (MDS) are heterogenous diseases which are characterized by ineffective ... more Myelodysplastic syndromes (MDS) are heterogenous diseases which are characterized by ineffective hematopoiesis with an increased risk of evolution to acute myeloid leukemia (AML). Diagnosis of MDS is mainly based on morphological findings and cytogenetical analyses. Since about 50% of MDS patients show a normal karyotype in conventional cytogenetics, the introduction of array based techniques in cancer research was logical and necessary to identify more subtle genomic changes such as cryptic deletions, regions of loss of heterozygosity and uniparental disomy. Moreover, since gene silencing by DNA methylation has been recognized as an important feature in the initiation and progression of human cancer, the development of high density methylation arrays provides the possibility to analyze the methylation status of a vast amount of CpG islands of promoters of (tumor suppressor) genes and genes with a so far unknown role in leukemogenesis.

Research paper thumbnail of Frequent pathway mutations of splicing machinery in myelodysplasia

Nature, 2011

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of mye... more Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent ( 45 to 85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 39-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

Research paper thumbnail of In acute promyelocytic leukemia (APL) low BAALC gene expression identifies a patient group with favorable overall survival and improved relapse free survival

Leukemia Research, 2013

We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leu... more We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leukemia (APL). At 10 years, the overall survival (OS) was 66% in all patients and 75% in patients who achieved a complete remission (CR). Patients in the BAALC(low) group showed an OS of 87% as compared to 60% in the BAALC(high) group (p=0.019). This difference was more pronounced in treatment responders (92% vs. 70%; p=0.035). In multivariate analyses low BAALC expression retained its prognostic relevance. In conclusion, BAALC expression analysis might be useful in further risk stratification in APL patients.

Research paper thumbnail of Depolarisation of the plasma membrane in the arsenic trioxide (As2O3)-and anti-CD95-induced apoptosis in myeloid cells

FEBS Letters, 2004

Depolarisation of the plasma membrane has been shown to be actively regulated during lymphocyte-a... more Depolarisation of the plasma membrane has been shown to be actively regulated during lymphocyte-apoptosis. Here, we present data about anti-Fas and As2O3 induced depolarisation of myeloid U-937 cells. Anti-Fas but not As2O3-induced depolarisation was significantly dependent on caspase-activation. Na+-fluxes contributed to the depolarisation in early stages of As2O3-induced apoptosis, whereas the membrane potential in late stages depended on Cl- -fluxes. Cl- -channels also played an important role in the induction of cell shrinkage in As2O3-induced apoptosis. However, none of these ions contributed significantly to anti-Fas induced depolarisation. This indicates the existence of different mechanisms for apoptotic plasma membrane depolarisation within one cell type.

Research paper thumbnail of Genome-wide DNA-mapping of CD34+ cells from patients with myelodysplastic syndrome using 500K SNP arrays identifies significant regions of deletion and uniparental disomy

Experimental Hematology, 2009

Objective. Identification of genomic lesions in progenitor cells of patients with myelodysplastic... more Objective. Identification of genomic lesions in progenitor cells of patients with myelodysplastic syndrome (MDS) could lead to the discovery of new disease-specific genes and may be of prognostic value. Materials and Methods. We carried out a genome-wide mapping of DNA from CD34+ cells of MDS patients with high-resolution 500K single nucleotide polymorphism arrays and a concomitant integration with global gene expression analysis. Thirteen MDS patients were analyzed. Results. Copy number and loss of heterozygosity analyses detected heterozygous deletions on chromosomes 2, 9, 13, 16, 17, and 20 ranging in size from 0.1 megabases (Mba) to 2.1 Mba. Additionally, numerous regions with significant uniparental disomy were detected. Integration of the genomic data with gene expression analysis showed that genes, which were downregulated at least 1.5-fold in regions of significant deletion and uniparental disomy were exclusively downregulated in those samples displaying the aberration. Genomics and gene expression data were confirmed by real-time polymerase chain reaction and variable number tandem repeat analysis. Conclusion. High-density genomic mapping of CD34+ bone marrow cells from patients with MDS identifies cryptic genetic lesions and offers new opportunities for the discovery of target genes in MDS by integration with gene expression analysis. Ó

Research paper thumbnail of The K+ channel openers diazoxide and NS1619 induce depolarization of mitochondria and have differential effects on cell Ca2+ in CD34+ cell line KG-1a

Experimental Hematology, 2003

Objective. Mitochondrial membrane potential (Dy m ) and intracellular Ca 2ϩ play a crucial role i... more Objective. Mitochondrial membrane potential (Dy m ) and intracellular Ca 2ϩ play a crucial role in growth and differentiation in hemopoiesis. Some potassium channel openers such as diazoxide have the capacity to elevate cytosolic Ca 2ϩ and depolarize mitochondria in cardiomyocytes. To clarify if such substances have effects on hemopoietic cells we investigated the commonly used opener of the mitoK ATP channel, diazoxide, and the opener of BK channels, NS1619, for their potential to depolarize mitochondria, elevate cytosolic Ca 2ϩ , and induce apoptosis in the hemopoietic CD34 ϩ cell line KG-1a. Methods. Fluorescent probes were used to investigate Dy m , free Ca 2ϩ , and apoptosis (JC-1, fluo-3-AM and annexin V-FITC) by flow cytometry. To measure Dy m with JC-1 in glycoprotein P ϩ cells we used an improved dye loading technique with verapamil. Results. NS1619 induced stronger dose-dependent mitochondrial depolarizations than diazoxide. Depolarization was independent from caspase activation and could also be induced when the driving force for K ϩ out of cells was near 0 mV. In Ca 2ϩ free solutions NS1619 induced stronger Ca 2ϩ elevations than diazoxide and elevated Ca 2ϩ also after Ca 2ϩ depletion of the endoplasmatic reticulum with caffeine. NS1619 did not enhance the Ca 2ϩ elevation induced by ionophores (CCCP, valinomycin) that depolarize mitochondria. Both agents were weak inducers of apoptosis. Conclusion. Diazoxide has similar effects in CD34 ϩ cells as described for muscle or nerve cells. In accordance to the single channel conductance of mitoK ATP and BK channels, NS1619 is a more potent inducer of mitochondrial depolarization than diazoxide. NS1619 releases Ca 2ϩ from an intracellular pool that is insensitive to caffeine but depends strongly on Dy m . Ć

Research paper thumbnail of Epigenetic dysregulation of GATA1 is involved in myelodysplastic syndromes dyserythropoiesis

European Journal of Haematology, 2012

Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This ... more Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This pathological hallmark is incompletely understood. Notch signaling has been linked to impaired erythropoietic and megakaryopoietic development of CD34+ progenitor cells, but its role in MDS is unclear. We have analyzed the transcriptional activity of Notch pathway elements and its association with the key erythroid factor globin transcription factor 1 (GATA1) and the apoptosis regulatory gene B-cell lymphoma-xl (BCLxl) in MDS. The methylation of GATA1 erythroid promoter CpG dinucleotides flanking cis-regulatory elements, including an N-box suppressor binding site for HES1 and a GATA-box binding site, was examined in normal and MDS erythropoiesis. We have generated a kinetic in vitro model of MDS erythropoiesis using CD34+ bone marrow cells from healthy donors (n = 7) and patients with MDS (low risk: RA/n = 6, RARS/n = 3; high risk: RAEB/n = 4, RAEB-T/n = 2). RNA expression of GATA1, BCLxl, DLK1, Notch1, HES1, and HERP2 was measured by real-time RT-PCR (qPCR). DNA methylation at seven CpG dinucleotides of the GATA1 gene promoter was quantitatively analyzed by pyrosequencing of bisulfite-treated genomic DNA at any specific time point. For the Notch pathway elements, no conclusive expression differences were found between MDS and normal erythropoiesis. But we found steadily up-regulated RNA expression of GATA1 and of BCLxl during late normal erythropoietic differentiation. In contrast, during MDS, erythropoiesis a loss of typical up-regulation of GATA1 and BCLxl was observed. Hypermethylation of CpG dinucleotides flanking the repressor HES1 binding site within the 5' region of GATA1 was detected particularly during late MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up-regulation. Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fails to be normally up-regulated during MDS erythropoiesis. The higher residual 5'-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggest a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS.

Research paper thumbnail of Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit

Cell Stem Cell, 2014

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in h... more Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

Research paper thumbnail of The role of mitochondrial targeting in arsenic trioxide-induced apoptosis in myeloid cell lines

British Journal of Haematology, 2004

Research paper thumbnail of Myelodysplastic syndromes: molecular pathogenesis and genomic changes

Annals of Hematology, 2008

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis presenting with pe... more Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis presenting with peripheral cytopenias in combination with a hyperplastic bone marrow and an increased risk of evolution to acute myeloid leukemia. The classification systems such as the WHO classification mainly rely on morphological criteria and are supplemented by the International Prognostic Scoring System which takes cytogenetical changes into consideration when determining the prognosis of MDS but wide intra-subtype variations do exist. The pathomechanisms causing primary MDS require further work. Development and progression of MDS is suggested to be a multistep alteration to hematopoietic stem cells. Different molecular alterations have been described, affecting genes involved in cell-cycle control, mitotic checkpoints, and growth factor receptors. Secondary signal proteins and transcription factors, which gives the cell a growth advantage over its normal counterpart, may be affected as well. The accumulation of such defects may finally cause the leukemic transformation of MDS.

Research paper thumbnail of Centrosome aberrations in bone marrow cells from patients with myelodysplastic syndromes correlate with chromosomal instability

Annals of Hematology, 2013

Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrati... more Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrations are a hallmark of cancer. Deregulation of centriole duplication leads to supernumerary centrosomes, sister chromatid missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 45% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. BM cells of 34 MDS patients were examined cytogenetically. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin to analyze the centrosomal status. Umbilical cord blood specimens and BM cells of healthy persons (n = 11 and n = 4) served as controls. In addition, the protein expression of the protease separase responsible for genetic stability was examined by western blot analysis. Centrosome abnormalities were detected in 10% (range, 4-17%) of cells of MDS samples, but in only 2% (range, 0-4%) of cells of healthy controls. Normal karyotypes were found in control cells and in BM cells of 16/34 MDS patients. The incidence of centrosomal alterations was higher in BM cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). Our results indicate that centrosome alterations are a common and early detectable feature in MDS patients and may contribute to the acquisition of chromosomal aberrations. We assume that centrosome defects could be involved in disease progression and may serve as a future prognostic marker.

Research paper thumbnail of Prognostic importance of expression of the Wilms’ tumor 1 gene in newly diagnosed acute promyelocytic leukemia

Leukemia & Lymphoma, 2015

Wilms&amp... more Wilms' tumor 1 gene (WT1) is known to be highly expressed in acute promyelocytic leukemia (APL) but information on its impact on prognosis is lacking. WT1 expression was analyzed in bone marrow samples of 79 patients with APL at initial diagnosis. Patients had a differing outcome according to their level of WT1 expression. In patients who achieved a complete remission (CR), low or high WT1 expression was significantly associated with inferior overall survival (OS) compared to intermediate WT1 expression (49% for WT1(high) vs. 63% for WT1(low) vs. 93% for WT1(int); p = 0.008). Moreover, there were significant differences in relapse-free survival (RFS) between the three expression groups (42% for WT1(high) vs. 63% for WT1(low) vs. 83% for WT1(int); p = 0.047). In multivariable analysis WT1 expression showed an independent prognostic impact on OS of responders to induction therapy. In conclusion, the level of WT1 expression can add prognostic information in APL risk stratification.

Research paper thumbnail of A molecular risk score integrating BAALC, ERG and WT1 expression levels for risk stratification in acute promyelocytic leukemia

Leukemia Research, 2015

To date risk stratification in acute promyelocytic leukemia (APL) is based on highly dynamic leuk... more To date risk stratification in acute promyelocytic leukemia (APL) is based on highly dynamic leukocyte and platelet counts only. To identify a more robust risk stratification model, a molecular risk score was developed based on expression levels of the genes BAALC, ERG and WT1. Hereby, the main focus was on prediction of relapse. The integrative risk score divided patients into two groups with highly significant differences in outcome. It discriminated a high risk group with a high incidence of relapse successfully from a low risk group with no APL-related events after achievement of first remission. Especially the concurrent presence of molecular risk factors showed to be a negative prognostic factor in APL. The molecular risk score might be a promising approach to guide monitoring of APL patients and therapeutic decisions in the future.

Research paper thumbnail of Telomere dynamics in patients with del (5q) MDS before and under treatment with lenalidomide

Leukemia Research, 2015

Myelodysplastic syndrome (MDS) associated with an acquired, isolated deletion of chromosome 5q (d... more Myelodysplastic syndrome (MDS) associated with an acquired, isolated deletion of chromosome 5q (del (5q) MDS), represent a clonal disorder of hematopoiesis and a clinically distinct entity of MDS. Treatment of del (5q) MDS with the drug lenalidomide has significantly improved quality of life leading to transfusion independence and complete cytogenetic response rates (CCR) in the majority of patients. Telomeres are located at the end of eukaryotic chromosomes and are linked to replicative history/potential as well as genetic (in) stability of hematopoietic stem cells. Here, we analyzed telomere length (TL) dynamics before and under lenalidomide treatment in the peripheral blood and/or bone marrow of del (5q) patients enrolled in the LEMON-5 study (NCT01081431). Hematopoietic cells from del (5q) MDS patients were characterized by significantly shortened TL compared to age-matched healthy controls. Telomere loss was more accelerated in patients with longer disease duration (>2 years) and more pronounced cytopenias. Sequential analysis under lenalidomide treatment revealed that previously shortened TL in peripheral blood cells was significantly "elongated" towards normal levels within the first six months suggesting a shift from clonal del (5q) cells towards normal hematopoiesis in lenalidomide treated MDS patients. Taken together our findings suggest that the development of the del (5q) clone is associated with accelerated telomere shortening at diagnosis. However, upon induction of CCR and reoccurrence of normal hematopoiesis, the lack of a persistent TL deficit argues against telomere-mediated genetic instability neither as a disease-promoting event of del (5q) MDS nor for lenalidomide mediated development of secondary primary malignancies of the hematopoietic system in responding patients.

Research paper thumbnail of Validation of cytogenetic risk groups according to International Prognostic Scoring Systems by peripheral blood CD34+FISH: results from a German diagnostic study in comparison with an international control group

Haematologica, 2015

International Prognostic Scoring Systems are used to determine the individual risk profile of mye... more International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%-20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34(+)) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization anal...

Research paper thumbnail of Molecular Changes in Myelodysplastic Syndrome

The Myelodysplastic Syndromes, 2011

Myelodysplastic syndromes (MDS) are heterogenous diseases which are characterized by ineffective ... more Myelodysplastic syndromes (MDS) are heterogenous diseases which are characterized by ineffective hematopoiesis with an increased risk of evolution to acute myeloid leukemia (AML). Diagnosis of MDS is mainly based on morphological findings and cytogenetical analyses. Since about 50% of MDS patients show a normal karyotype in conventional cytogenetics, the introduction of array based techniques in cancer research was logical and necessary to identify more subtle genomic changes such as cryptic deletions, regions of loss of heterozygosity and uniparental disomy. Moreover, since gene silencing by DNA methylation has been recognized as an important feature in the initiation and progression of human cancer, the development of high density methylation arrays provides the possibility to analyze the methylation status of a vast amount of CpG islands of promoters of (tumor suppressor) genes and genes with a so far unknown role in leukemogenesis.

Research paper thumbnail of Frequent pathway mutations of splicing machinery in myelodysplasia

Nature, 2011

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of mye... more Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent ( 45 to 85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 39-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

Research paper thumbnail of In acute promyelocytic leukemia (APL) low BAALC gene expression identifies a patient group with favorable overall survival and improved relapse free survival

Leukemia Research, 2013

We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leu... more We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leukemia (APL). At 10 years, the overall survival (OS) was 66% in all patients and 75% in patients who achieved a complete remission (CR). Patients in the BAALC(low) group showed an OS of 87% as compared to 60% in the BAALC(high) group (p=0.019). This difference was more pronounced in treatment responders (92% vs. 70%; p=0.035). In multivariate analyses low BAALC expression retained its prognostic relevance. In conclusion, BAALC expression analysis might be useful in further risk stratification in APL patients.

Research paper thumbnail of Depolarisation of the plasma membrane in the arsenic trioxide (As2O3)-and anti-CD95-induced apoptosis in myeloid cells

FEBS Letters, 2004

Depolarisation of the plasma membrane has been shown to be actively regulated during lymphocyte-a... more Depolarisation of the plasma membrane has been shown to be actively regulated during lymphocyte-apoptosis. Here, we present data about anti-Fas and As2O3 induced depolarisation of myeloid U-937 cells. Anti-Fas but not As2O3-induced depolarisation was significantly dependent on caspase-activation. Na+-fluxes contributed to the depolarisation in early stages of As2O3-induced apoptosis, whereas the membrane potential in late stages depended on Cl- -fluxes. Cl- -channels also played an important role in the induction of cell shrinkage in As2O3-induced apoptosis. However, none of these ions contributed significantly to anti-Fas induced depolarisation. This indicates the existence of different mechanisms for apoptotic plasma membrane depolarisation within one cell type.

Research paper thumbnail of Genome-wide DNA-mapping of CD34+ cells from patients with myelodysplastic syndrome using 500K SNP arrays identifies significant regions of deletion and uniparental disomy

Experimental Hematology, 2009

Objective. Identification of genomic lesions in progenitor cells of patients with myelodysplastic... more Objective. Identification of genomic lesions in progenitor cells of patients with myelodysplastic syndrome (MDS) could lead to the discovery of new disease-specific genes and may be of prognostic value. Materials and Methods. We carried out a genome-wide mapping of DNA from CD34+ cells of MDS patients with high-resolution 500K single nucleotide polymorphism arrays and a concomitant integration with global gene expression analysis. Thirteen MDS patients were analyzed. Results. Copy number and loss of heterozygosity analyses detected heterozygous deletions on chromosomes 2, 9, 13, 16, 17, and 20 ranging in size from 0.1 megabases (Mba) to 2.1 Mba. Additionally, numerous regions with significant uniparental disomy were detected. Integration of the genomic data with gene expression analysis showed that genes, which were downregulated at least 1.5-fold in regions of significant deletion and uniparental disomy were exclusively downregulated in those samples displaying the aberration. Genomics and gene expression data were confirmed by real-time polymerase chain reaction and variable number tandem repeat analysis. Conclusion. High-density genomic mapping of CD34+ bone marrow cells from patients with MDS identifies cryptic genetic lesions and offers new opportunities for the discovery of target genes in MDS by integration with gene expression analysis. Ó

Research paper thumbnail of The K+ channel openers diazoxide and NS1619 induce depolarization of mitochondria and have differential effects on cell Ca2+ in CD34+ cell line KG-1a

Experimental Hematology, 2003

Objective. Mitochondrial membrane potential (Dy m ) and intracellular Ca 2ϩ play a crucial role i... more Objective. Mitochondrial membrane potential (Dy m ) and intracellular Ca 2ϩ play a crucial role in growth and differentiation in hemopoiesis. Some potassium channel openers such as diazoxide have the capacity to elevate cytosolic Ca 2ϩ and depolarize mitochondria in cardiomyocytes. To clarify if such substances have effects on hemopoietic cells we investigated the commonly used opener of the mitoK ATP channel, diazoxide, and the opener of BK channels, NS1619, for their potential to depolarize mitochondria, elevate cytosolic Ca 2ϩ , and induce apoptosis in the hemopoietic CD34 ϩ cell line KG-1a. Methods. Fluorescent probes were used to investigate Dy m , free Ca 2ϩ , and apoptosis (JC-1, fluo-3-AM and annexin V-FITC) by flow cytometry. To measure Dy m with JC-1 in glycoprotein P ϩ cells we used an improved dye loading technique with verapamil. Results. NS1619 induced stronger dose-dependent mitochondrial depolarizations than diazoxide. Depolarization was independent from caspase activation and could also be induced when the driving force for K ϩ out of cells was near 0 mV. In Ca 2ϩ free solutions NS1619 induced stronger Ca 2ϩ elevations than diazoxide and elevated Ca 2ϩ also after Ca 2ϩ depletion of the endoplasmatic reticulum with caffeine. NS1619 did not enhance the Ca 2ϩ elevation induced by ionophores (CCCP, valinomycin) that depolarize mitochondria. Both agents were weak inducers of apoptosis. Conclusion. Diazoxide has similar effects in CD34 ϩ cells as described for muscle or nerve cells. In accordance to the single channel conductance of mitoK ATP and BK channels, NS1619 is a more potent inducer of mitochondrial depolarization than diazoxide. NS1619 releases Ca 2ϩ from an intracellular pool that is insensitive to caffeine but depends strongly on Dy m . Ć

Research paper thumbnail of Epigenetic dysregulation of GATA1 is involved in myelodysplastic syndromes dyserythropoiesis

European Journal of Haematology, 2012

Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This ... more Myelodysplastic syndromes (MDS) are characterized by dyserythropoiesis resulting in anemia. This pathological hallmark is incompletely understood. Notch signaling has been linked to impaired erythropoietic and megakaryopoietic development of CD34+ progenitor cells, but its role in MDS is unclear. We have analyzed the transcriptional activity of Notch pathway elements and its association with the key erythroid factor globin transcription factor 1 (GATA1) and the apoptosis regulatory gene B-cell lymphoma-xl (BCLxl) in MDS. The methylation of GATA1 erythroid promoter CpG dinucleotides flanking cis-regulatory elements, including an N-box suppressor binding site for HES1 and a GATA-box binding site, was examined in normal and MDS erythropoiesis. We have generated a kinetic in vitro model of MDS erythropoiesis using CD34+ bone marrow cells from healthy donors (n = 7) and patients with MDS (low risk: RA/n = 6, RARS/n = 3; high risk: RAEB/n = 4, RAEB-T/n = 2). RNA expression of GATA1, BCLxl, DLK1, Notch1, HES1, and HERP2 was measured by real-time RT-PCR (qPCR). DNA methylation at seven CpG dinucleotides of the GATA1 gene promoter was quantitatively analyzed by pyrosequencing of bisulfite-treated genomic DNA at any specific time point. For the Notch pathway elements, no conclusive expression differences were found between MDS and normal erythropoiesis. But we found steadily up-regulated RNA expression of GATA1 and of BCLxl during late normal erythropoietic differentiation. In contrast, during MDS, erythropoiesis a loss of typical up-regulation of GATA1 and BCLxl was observed. Hypermethylation of CpG dinucleotides flanking the repressor HES1 binding site within the 5' region of GATA1 was detected particularly during late MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up-regulation. Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fails to be normally up-regulated during MDS erythropoiesis. The higher residual 5'-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggest a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS.

Research paper thumbnail of Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit

Cell Stem Cell, 2014

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in h... more Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

Research paper thumbnail of The role of mitochondrial targeting in arsenic trioxide-induced apoptosis in myeloid cell lines

British Journal of Haematology, 2004

Research paper thumbnail of Myelodysplastic syndromes: molecular pathogenesis and genomic changes

Annals of Hematology, 2008

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis presenting with pe... more Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis presenting with peripheral cytopenias in combination with a hyperplastic bone marrow and an increased risk of evolution to acute myeloid leukemia. The classification systems such as the WHO classification mainly rely on morphological criteria and are supplemented by the International Prognostic Scoring System which takes cytogenetical changes into consideration when determining the prognosis of MDS but wide intra-subtype variations do exist. The pathomechanisms causing primary MDS require further work. Development and progression of MDS is suggested to be a multistep alteration to hematopoietic stem cells. Different molecular alterations have been described, affecting genes involved in cell-cycle control, mitotic checkpoints, and growth factor receptors. Secondary signal proteins and transcription factors, which gives the cell a growth advantage over its normal counterpart, may be affected as well. The accumulation of such defects may finally cause the leukemic transformation of MDS.

Research paper thumbnail of Centrosome aberrations in bone marrow cells from patients with myelodysplastic syndromes correlate with chromosomal instability

Annals of Hematology, 2013

Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrati... more Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrations are a hallmark of cancer. Deregulation of centriole duplication leads to supernumerary centrosomes, sister chromatid missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 45% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. BM cells of 34 MDS patients were examined cytogenetically. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin to analyze the centrosomal status. Umbilical cord blood specimens and BM cells of healthy persons (n = 11 and n = 4) served as controls. In addition, the protein expression of the protease separase responsible for genetic stability was examined by western blot analysis. Centrosome abnormalities were detected in 10% (range, 4-17%) of cells of MDS samples, but in only 2% (range, 0-4%) of cells of healthy controls. Normal karyotypes were found in control cells and in BM cells of 16/34 MDS patients. The incidence of centrosomal alterations was higher in BM cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). Our results indicate that centrosome alterations are a common and early detectable feature in MDS patients and may contribute to the acquisition of chromosomal aberrations. We assume that centrosome defects could be involved in disease progression and may serve as a future prognostic marker.

Research paper thumbnail of Prognostic importance of expression of the Wilms’ tumor 1 gene in newly diagnosed acute promyelocytic leukemia

Leukemia & Lymphoma, 2015

Wilms&amp... more Wilms' tumor 1 gene (WT1) is known to be highly expressed in acute promyelocytic leukemia (APL) but information on its impact on prognosis is lacking. WT1 expression was analyzed in bone marrow samples of 79 patients with APL at initial diagnosis. Patients had a differing outcome according to their level of WT1 expression. In patients who achieved a complete remission (CR), low or high WT1 expression was significantly associated with inferior overall survival (OS) compared to intermediate WT1 expression (49% for WT1(high) vs. 63% for WT1(low) vs. 93% for WT1(int); p = 0.008). Moreover, there were significant differences in relapse-free survival (RFS) between the three expression groups (42% for WT1(high) vs. 63% for WT1(low) vs. 83% for WT1(int); p = 0.047). In multivariable analysis WT1 expression showed an independent prognostic impact on OS of responders to induction therapy. In conclusion, the level of WT1 expression can add prognostic information in APL risk stratification.