- Email: [email protected]
C010 Differentially expressed genes of CD34+ between patients with early and advanced myelodysplastic syndrome
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Oral Communications
may increase sensitivity, especially in patients with low grade MDS. A validated aCGH chip to detect the genomic regions implicated in the myeloid disorders has the potential to improve clinical assessment, prognosis, and possibly therapy decisions for patients with MDS. This feasibility study shows the potential of this technology to identify recurring imbalances with the identification of new regions that may prove to be significant in disease progression or transformation to AML. Novel findings may lead to new markers for prognosis and potential treatment targets.
C009 Pyrosequencing reveals the quantity of p15INK4b methylation has correlation with cytopenia, marrow blast percentage and survival in myelodysplastic syndromes D. Lee1 ° , M. Kim1 , B. Oh2 , H. Lee1 , H. Park1 , C. She1 , T. Kim2 , S. Hwang1 , I. Yang3 , S. Yoon4 , J. Yoon5 . 1 Department of Laboratory Medicine, Seoul National University Hospital, Seoul, South Korea; 2 Cancer Research Institute, Seoul National University College of Medicine, South Korea; 3 Organic and Bio-analysis group, Korea Research Institute of Standards and Science, South Korea; 4 Department of Internal Medicine, Seoul National University Hospital, South Korea; 5 Department of Laboratory Medicine, Seoul National University Boramae Hospital, South Korea *E-mail: [email protected] Introduction: The density of methylation of the CpG islands is associated with the repression of gene transcription. We performed Pyrosequencing for the quantitation of p15INK4b promoter methylation to elucidate it and the disease heterogeneity in MDS. Materials and Methods: Pyrosequencing was performed with bone marrow cells of 74 de novo MDS patients and the correlations between pyrosequencing results and variables in IPSS, IPSS risk groups were evaluated. Two parameters were: methylation index (MtI) − the percentage of methylated CpG out of 11 CpG sites; methylation level (MtL) − the mean of methylated cytosine percentage in 11 CpG sites. Result: Both the mean MtI and mean MtL were higher in high-grade MDS (5−20% bone marrow blasts) than in low-grade MDS (less than 5% blasts) (p0.031, MtI). Both the mean MtI and the mean MtL were high in groups with anemia, neutropenia or thrombocytopenia with statistical significance in thrombocytopenia group (p0.006 and 0.030, respectively). Patients with cytopenias in 2−3 lineages showed higher mean MtI and mean MtL than those with cytopenias in 0−1 lineage (p0.055 and 0.048, respectively). Patients with lower than mean MtI or MtF showed better survival than those with higher than mean MtI or MtF in univariate analysis (p0.026 and 0.030, respectively).
Conclusion: The quantity of p15INK4b methylation determines the prognosis of MDS patients in correlation with other prognostic factors such as marrow blast percentage or cytopenia, even though the quantity of p15INK4b methylation is not an independent prognostic factor.
C010 Differentially expressed genes of CD34+ between patients with early and advanced myelodysplastic syndrome M. Belickova1 ° , A. Vasikova1 , E. Budinska2 , D. Mikulenkova1 , K. Michalova1 , J. Cermak1 . 1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic; 2 Institute of Biostatistics and Analyses at the Faculty of Medicine of the Masaryk University, Czech Republic *E-mail: [email protected] Purpose: Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal disorders with ineffective hematopoiesis that is characterized by dysplasia and peripheral cytopenia of one or more cell lineages. The defect of hematopoietic stem cell in MDS is not well characterized and it is assumed to be a multistep process. To expand the understanding of genetic defects responsible for the evolution of early MDS to advanced stages and AML, we performed gene expression analysis in MDS patients. Methods: We compared gene expression profiles of CD34+ cells obtained from 15 patients with early MDS (RA, RCMD), 15 patients with advanced MDS (RAEB 2) and 7 controls. Using HumanRef-8 v2 Expression BeadChips (Illumina) we analyzed expression levels of more than 23 000 transcripts in all tested sample. Normalized data (lumi package R software) were filtered by detection p-value <0.01. Filtered set of 9811 genes was tested for differential expression between clinical groups and control group. Our results were confirmed by real-time quantitative PCR for several genes (TaqMan Gene Expression Assays). Results and Conclusions: Based on statistical analysis, 550 significantly differentially expressed genes between two groups of patients were detected. Hierarchical clustering analysis (Sperman correlation) clearly distinguished early MDS patient from those with advanced MDS. Out of these, 234 genes showed higher expression levels in early MDS patients compared to advanced MDS. Using DAVID database we identified the most significant gene ontology categories for these genes: cell cycle process (33 genes), DNA repair (17genes) and response to stress (34). Further, 3 deregulated pathways were found: DNA Polymerase (POLA2, POLQ, PRIM1, POLE2), p53 signaling pathway (CHEK1, CCNB1, CCNB2, CDC2, STEAP3), and cell cycle (e.g. CDC25A, BUB1, TFDP1, E2F2). We showed reduced expression in early MDS patients versus advanced MDS for 316 genes and their functional annotation
2. Molecular Mechanisms and Pathophysiology resulted in identification of following biological processes: cell adhesion (21genes), cell proliferation (18 genes) angiogenesis (6 genes) regulation of small GTPase mediated signal transduction (8genes) and blood vessel morphogenesis (6). DAVID database showed a few pathways in this set of genes: gap junction (e.g. PRKCB1, ITPR1, ADCY6, PDGFC), hematopoietic cell lineage (e.g. IL11RA, FLT3, ITGA5, ANPEP, CD55), leukocyte transendothelial migration (e.g. PRKCB1, RAPGEF3, F11R, ITGB2, CD99), and cell adhesion molecules (ICAM3, CLDN15, NRXN2). We focused on the genes showing the most distinctive expression change (lower expression in advanced than in early MDS): LEF1, VPREB1, VPREB3, IRF8, HES6. Lef-1 is a major transcriptional factor in TGF-b and Wnt signaling cascade, and its higher expression levels in patients with RA compared to those with RAEB 2 was previously described (Pellagatti, Blood. 2006; 108:337−45). VPREB1 and VPREB3 are known to be involved in B-lymphocyte development, as well as Lef-1 mentioned above. (Sternberg A, Blood. 2005; 106:2982−91). IRF8 regulates the proliferation and differentiation of hematopoietic progenitor cells and also it was identified as a functional tumor suppressor, which is frequently silenced by epigenetic mechanism. Hes6 is a transcriptional factor that functions in the differentiation of pluripotent progenitor cells and inhibition of cell proliferation. (Eun B, J Biol Chem. 2008; 283:5939−49). This gene was up-regulated in early MDS and down regulated in advanced MDS. The abundant expression in advanced MDS than early MDS was found for STAB1, ANGPT1, NRXN2, PTHR2 and MAMDC2. STAB1 may function in angiogenesis and cell adhesion. ANGPT1 is a protein with important roles in vascular development and angiogenesis and hits higher expression was observed in AML. Direct association between expression levels of the rest of genes and disease progression has not been reported yet. In this study we report plenty of genes with significantly dysregulated expression, which can be responsible for progression disease. Development and progression of MDS to AML involve many mutations of cell-cycle, DNA repair, cell adhesion and angiogenesis genes. We observed increased level of cell-cycle and DNA repair genes in early MDS patients and reversely increased level of cell adhesion and angiogenesis genes in advanced MDS patients. This study was supported by the grant IGA NR9235.
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2. Molecular Mechanisms and Pathophysiology C011 Gene methylation, telomere length and activity of telomerase reflect the disease activity in MDS patients and may help to assess an individual risk of patients H. Cechova1 ° , R. Stemberkova1 , S. Vcelikova1 , J. Cermak2 , M. Stankova1 , Z. Zemanova3 , K. Michalova3 . 1 Institute of Hematology and Blood Transfusion, National Reference for DNA diagnostics, Prague, Prague, Czech Republic; 2 Institute of Hematology and Blood Transfusion, Clinical Ward, Prague, Czech Republic; 3 1st School of medicine of Charles University, Prague, Czech Republic *E-mail: [email protected] Background: Knowledge of telomere-telomerase complex and epigenetic de novo methylation of gene CDKN2B may bring an important insight into molecular background of malignant transformation. Methods: Level of methylation DNA was quantified by methylation specific PCR after sodium bisulphate modification. Methylation status was dedicated as Methylation Indices (MI: 0.0−1.0). Telomere length was determined as terminal restriction fragment (TRF index in kbp), activity of telomerase by Quantitative TRAP assay. Results: We analysed 110 patients with different subtypes of MDS. Changes in methylation intensity were observed in 64% of untreated MDS patients. Significant difference was found between early and advanced forms with excess of blasts. In most cases an aberrant methylation correlated with increased telomerase activity and with shortening of telomere length. In patient with RAEB2 the value of MI at the diagnosis was 0.37 (TRF=7.62 kbp). Decrease of MI from 0.25 to 0.04 together with prolongation of telomere length (TRF=10.5 kbp) was observed after 3 months of treatment with Decitabine. After 6 months of the treatment a patient achieved a partial remission of the disease with MI=0.20 and TRF=9.63 kbp. In another patient with RCMD an increase in MI value from 0.00 to 0.39 during 6 months correlated with elevation of telomerase activity from 0 to 0.0534 and with reflected a subsequent progression to RAEB1. Conclusions: Molecular changes in gene methylation were compared with the telomere length, telomerase activity and clinical parameters. These results showed correlations between individual markers, reflecting the activity of the disease. Investigation of MI, TRF and telomerase activity may contribute to more precise estimation of an individual patient’s risk and thus for decision of an optimal treatment strategy. Granted by NR-9235−3
Oral Communications
may increase sensitivity, especially in patients with low grade MDS. A validated aCGH chip to detect the genomic regions implicated in the myeloid disorders has the potential to improve clinical assessment, prognosis, and possibly therapy decisions for patients with MDS. This feasibility study shows the potential of this technology to identify recurring imbalances with the identification of new regions that may prove to be significant in disease progression or transformation to AML. Novel findings may lead to new markers for prognosis and potential treatment targets.
C009 Pyrosequencing reveals the quantity of p15INK4b methylation has correlation with cytopenia, marrow blast percentage and survival in myelodysplastic syndromes D. Lee1 ° , M. Kim1 , B. Oh2 , H. Lee1 , H. Park1 , C. She1 , T. Kim2 , S. Hwang1 , I. Yang3 , S. Yoon4 , J. Yoon5 . 1 Department of Laboratory Medicine, Seoul National University Hospital, Seoul, South Korea; 2 Cancer Research Institute, Seoul National University College of Medicine, South Korea; 3 Organic and Bio-analysis group, Korea Research Institute of Standards and Science, South Korea; 4 Department of Internal Medicine, Seoul National University Hospital, South Korea; 5 Department of Laboratory Medicine, Seoul National University Boramae Hospital, South Korea *E-mail: [email protected] Introduction: The density of methylation of the CpG islands is associated with the repression of gene transcription. We performed Pyrosequencing for the quantitation of p15INK4b promoter methylation to elucidate it and the disease heterogeneity in MDS. Materials and Methods: Pyrosequencing was performed with bone marrow cells of 74 de novo MDS patients and the correlations between pyrosequencing results and variables in IPSS, IPSS risk groups were evaluated. Two parameters were: methylation index (MtI) − the percentage of methylated CpG out of 11 CpG sites; methylation level (MtL) − the mean of methylated cytosine percentage in 11 CpG sites. Result: Both the mean MtI and mean MtL were higher in high-grade MDS (5−20% bone marrow blasts) than in low-grade MDS (less than 5% blasts) (p0.031, MtI). Both the mean MtI and the mean MtL were high in groups with anemia, neutropenia or thrombocytopenia with statistical significance in thrombocytopenia group (p0.006 and 0.030, respectively). Patients with cytopenias in 2−3 lineages showed higher mean MtI and mean MtL than those with cytopenias in 0−1 lineage (p0.055 and 0.048, respectively). Patients with lower than mean MtI or MtF showed better survival than those with higher than mean MtI or MtF in univariate analysis (p0.026 and 0.030, respectively).
Conclusion: The quantity of p15INK4b methylation determines the prognosis of MDS patients in correlation with other prognostic factors such as marrow blast percentage or cytopenia, even though the quantity of p15INK4b methylation is not an independent prognostic factor.
C010 Differentially expressed genes of CD34+ between patients with early and advanced myelodysplastic syndrome M. Belickova1 ° , A. Vasikova1 , E. Budinska2 , D. Mikulenkova1 , K. Michalova1 , J. Cermak1 . 1 Institute of Hematology and Blood Transfusion, Prague, Czech Republic; 2 Institute of Biostatistics and Analyses at the Faculty of Medicine of the Masaryk University, Czech Republic *E-mail: [email protected] Purpose: Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal disorders with ineffective hematopoiesis that is characterized by dysplasia and peripheral cytopenia of one or more cell lineages. The defect of hematopoietic stem cell in MDS is not well characterized and it is assumed to be a multistep process. To expand the understanding of genetic defects responsible for the evolution of early MDS to advanced stages and AML, we performed gene expression analysis in MDS patients. Methods: We compared gene expression profiles of CD34+ cells obtained from 15 patients with early MDS (RA, RCMD), 15 patients with advanced MDS (RAEB 2) and 7 controls. Using HumanRef-8 v2 Expression BeadChips (Illumina) we analyzed expression levels of more than 23 000 transcripts in all tested sample. Normalized data (lumi package R software) were filtered by detection p-value <0.01. Filtered set of 9811 genes was tested for differential expression between clinical groups and control group. Our results were confirmed by real-time quantitative PCR for several genes (TaqMan Gene Expression Assays). Results and Conclusions: Based on statistical analysis, 550 significantly differentially expressed genes between two groups of patients were detected. Hierarchical clustering analysis (Sperman correlation) clearly distinguished early MDS patient from those with advanced MDS. Out of these, 234 genes showed higher expression levels in early MDS patients compared to advanced MDS. Using DAVID database we identified the most significant gene ontology categories for these genes: cell cycle process (33 genes), DNA repair (17genes) and response to stress (34). Further, 3 deregulated pathways were found: DNA Polymerase (POLA2, POLQ, PRIM1, POLE2), p53 signaling pathway (CHEK1, CCNB1, CCNB2, CDC2, STEAP3), and cell cycle (e.g. CDC25A, BUB1, TFDP1, E2F2). We showed reduced expression in early MDS patients versus advanced MDS for 316 genes and their functional annotation
2. Molecular Mechanisms and Pathophysiology resulted in identification of following biological processes: cell adhesion (21genes), cell proliferation (18 genes) angiogenesis (6 genes) regulation of small GTPase mediated signal transduction (8genes) and blood vessel morphogenesis (6). DAVID database showed a few pathways in this set of genes: gap junction (e.g. PRKCB1, ITPR1, ADCY6, PDGFC), hematopoietic cell lineage (e.g. IL11RA, FLT3, ITGA5, ANPEP, CD55), leukocyte transendothelial migration (e.g. PRKCB1, RAPGEF3, F11R, ITGB2, CD99), and cell adhesion molecules (ICAM3, CLDN15, NRXN2). We focused on the genes showing the most distinctive expression change (lower expression in advanced than in early MDS): LEF1, VPREB1, VPREB3, IRF8, HES6. Lef-1 is a major transcriptional factor in TGF-b and Wnt signaling cascade, and its higher expression levels in patients with RA compared to those with RAEB 2 was previously described (Pellagatti, Blood. 2006; 108:337−45). VPREB1 and VPREB3 are known to be involved in B-lymphocyte development, as well as Lef-1 mentioned above. (Sternberg A, Blood. 2005; 106:2982−91). IRF8 regulates the proliferation and differentiation of hematopoietic progenitor cells and also it was identified as a functional tumor suppressor, which is frequently silenced by epigenetic mechanism. Hes6 is a transcriptional factor that functions in the differentiation of pluripotent progenitor cells and inhibition of cell proliferation. (Eun B, J Biol Chem. 2008; 283:5939−49). This gene was up-regulated in early MDS and down regulated in advanced MDS. The abundant expression in advanced MDS than early MDS was found for STAB1, ANGPT1, NRXN2, PTHR2 and MAMDC2. STAB1 may function in angiogenesis and cell adhesion. ANGPT1 is a protein with important roles in vascular development and angiogenesis and hits higher expression was observed in AML. Direct association between expression levels of the rest of genes and disease progression has not been reported yet. In this study we report plenty of genes with significantly dysregulated expression, which can be responsible for progression disease. Development and progression of MDS to AML involve many mutations of cell-cycle, DNA repair, cell adhesion and angiogenesis genes. We observed increased level of cell-cycle and DNA repair genes in early MDS patients and reversely increased level of cell adhesion and angiogenesis genes in advanced MDS patients. This study was supported by the grant IGA NR9235.
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2. Molecular Mechanisms and Pathophysiology C011 Gene methylation, telomere length and activity of telomerase reflect the disease activity in MDS patients and may help to assess an individual risk of patients H. Cechova1 ° , R. Stemberkova1 , S. Vcelikova1 , J. Cermak2 , M. Stankova1 , Z. Zemanova3 , K. Michalova3 . 1 Institute of Hematology and Blood Transfusion, National Reference for DNA diagnostics, Prague, Prague, Czech Republic; 2 Institute of Hematology and Blood Transfusion, Clinical Ward, Prague, Czech Republic; 3 1st School of medicine of Charles University, Prague, Czech Republic *E-mail: [email protected] Background: Knowledge of telomere-telomerase complex and epigenetic de novo methylation of gene CDKN2B may bring an important insight into molecular background of malignant transformation. Methods: Level of methylation DNA was quantified by methylation specific PCR after sodium bisulphate modification. Methylation status was dedicated as Methylation Indices (MI: 0.0−1.0). Telomere length was determined as terminal restriction fragment (TRF index in kbp), activity of telomerase by Quantitative TRAP assay. Results: We analysed 110 patients with different subtypes of MDS. Changes in methylation intensity were observed in 64% of untreated MDS patients. Significant difference was found between early and advanced forms with excess of blasts. In most cases an aberrant methylation correlated with increased telomerase activity and with shortening of telomere length. In patient with RAEB2 the value of MI at the diagnosis was 0.37 (TRF=7.62 kbp). Decrease of MI from 0.25 to 0.04 together with prolongation of telomere length (TRF=10.5 kbp) was observed after 3 months of treatment with Decitabine. After 6 months of the treatment a patient achieved a partial remission of the disease with MI=0.20 and TRF=9.63 kbp. In another patient with RCMD an increase in MI value from 0.00 to 0.39 during 6 months correlated with elevation of telomerase activity from 0 to 0.0534 and with reflected a subsequent progression to RAEB1. Conclusions: Molecular changes in gene methylation were compared with the telomere length, telomerase activity and clinical parameters. These results showed correlations between individual markers, reflecting the activity of the disease. Investigation of MI, TRF and telomerase activity may contribute to more precise estimation of an individual patient’s risk and thus for decision of an optimal treatment strategy. Granted by NR-9235−3