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  • 1
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    Genomic CDKN2A/2B deletions in adult Ph+ ALL are adverse despite allogeneic stem cell transplantation
    American Society of Hematology ; 2018
    In:  Blood Vol. 131, No. 13 ( 2018-03-29), p. 1464-1475
    Details
    In: Blood, American Society of Hematology, Vol. 131, No. 13 ( 2018-03-29), p. 1464-1475
    Abstract: Genomic deletions of CDKN2A/2B are a new independent prognostic risk factor in adult Ph+ ALL.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2017-07-796862
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
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  • 2
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    Significant Engraftment of Immature Hematopoietic Cells From Patients with Low Risk Myelodysplastic Syndromes (MDS) in Immunodeficient Mice
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 1694-1694
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 1694-1694
    Abstract: Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.1694.1694
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 3
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    Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit
    Elsevier BV ; 2014
    In:  Cell Stem Cell Vol. 14, No. 6 ( 2014-06), p. 824-837
    Details
    In: Cell Stem Cell, Elsevier BV, Vol. 14, No. 6 ( 2014-06), p. 824-837
    Type of Medium: Online Resource
    ISSN: 1934-5909
    URL: Article
    DOI: 10.1016/j.stem.2014.02.014
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2014
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  • 4
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    Next Generation RNA Sequencing of Acute Promyelocytic Leukemia (APL) Identifies Novel Long Non Coding RNAs Including New Variants of MIR181A1HG That Are Differentially Expressed during Myeloid Differentiation
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 1031-1031
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1031-1031
    Abstract: Introduction: Recently we identified a recurrent acquired genomic deletion on chromosome 1q as a potential new marker in approximately 14% of APL patients predicting a significantly increased risk of relapse (Nowak D et al., Genes Chromosomes and Cancer 2012). The deleted region contains the coding sequences for the microRNAs hsa-mir-181a1 and hsa-mir-181b1, which have been implicated as prognostic factors in Acute Myeloid Leukemia (AML) and a corresponding host gene (MIR181A1HG). To elucidate biologic mechanisms associated with the described genomic deletion we performed targeted sequencing of the affected region and RNA sequencing of APL samples carrying the deletion versus samples not carrying the deletion with subsequent validation of novel variants of MIR181A1HG. Methods: Explorative sequencing of genomic DNA in the chromosomal subband 1q31.3, pos. 197073900-197196158 (hg18) was performed using the amplicon sequencing workflow of the Roche 454 platform sequencing 5000 bp fragments tiling a region of approximately 120 kb on n=3 APL samples. Corresponding patient samples from molecular remission were used as germline controls. Whole transcriptome sequencing of poly-A enriched RNA was performed on n=6 samples of bone marrow blasts of APL patients either carrying a deletion of the mir181a1/b1 coding region (n=3) or not carrying a deletion (n=3). RNA Sequencing was performed using the HiSeq2000 platform. Data analysis was carried out using Bowtie vers. 2.2.30, TopHat vers. 2.0.12 for alignment and mapping and the Cufflinks package vers. 2.2.1 for transcriptome assembly and expression analysis all using default settings and hg19 as reference genome. Validation of newly identified variants and differential expression of MIR181A1HG was carried out by RACE PCR and qRT-PCR on cDNA from primary leukemic blasts of APL patients (n=45), CD34+ cells from healthy donors (n=29). In vitro differentiation assays with concomitant gene expression analysis of MIR181A1HG variants were performed with CD34+ cells from healthy donors. Results: Genomic sequencing of the recurrently deleted region revealed no somatically acquired mutations in the analyzed APL samples. Differential gene expression analysis using FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) inferred from RNA sequencing data of APL samples carrying a genomic deletion of 1q31.3 versus non-deleted samples identified n=58 genes significantly downregulated in deleted samples and n=31 upregulated genes. Interestingly, among the differentially regulated genes, BAALC, a factor recently shown to be prognostically relevant in APL was significantly upregulated 13 fold in the unfavourable group of samples with 1q31.3 deletions. Furthermore, RNA sequencing revealed numerous new isoforms of known transcripts as well as novel long non-conding RNA (lncRNA) sequences. Among these were a total of 6 new transcript variants of the MIR181A1HG gene in the recurrently deleted region on chromosome 1q31.3. One novel 5600bp lncRNA covering the coding regions for the hsa-mir-181a1/b1 was 24 fold overexpressed in samples carrying the recurrent 1q31.3 deletions. Expression analysis of MIR181A1HG in blasts of APL patients, CD34+ cells, unselected bone marrow cells and granulocytes of healthy donors revealed significantly elevated levels of MIR181A1HG in APL cells as compared to healthy CD34+ cells and almost absent expression in unselected bone marrow and granulocytes. This indicated a possible role for MIR181A1HG in APL blasts and hematopoietic stem cells. Subsequent in vitro differentiation experiments of primary healthy CD34+ cells showed that MIR181A1HG is downregulated 7 fold within 14 days of cytokine induced myeloid differentiation. Furthermore, MIR181A1HG was downregulated 5 fold during ATRA induced differentiation of NB4 cells. Conclusion: RNA sequencing of APL cells demonstrated numerous novel uncharacterized lncRNAs whose expression is associated with clinical risk and which merit further investigation. Identification of novel isoforms of MIR181A1HG, which are highly expressed in APL blasts and purified CD34+ cells suggest a potential role for this lncRNA in hematopoietic stem cells and response to ATRA induced differentiation of APL cells. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.1031.1031
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 5
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    Clinical Impact of TP53 Mutations in Patients with MDS and Isolated Deletion 5(q) Treated with Lenalidomid: Results from the German Prospective Le-Mon-5 Trial
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 1920-1920
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1920-1920
    Abstract: Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53 have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q). Methods: Le-Mon-5 is a trial of Len in patients with MDS with isolated 5q abnormality, a blast count of 〈 5% in the bone marrow, a platelet count 〉 50.000/µl and absolute neutrophil counts of 〉 500/µl in the peripheral blood. Patients were treated with the standard dose of 10 mg Len for 21 days q28 days. The primary endpoint was safety. Secondary endpoints included response according to IWG criteria (2000), time to response, duration of transfusion independency and incidence and time to AML-transformation, respectively. All patients gave written informed consent to the trial including additional molecular genetic analyses. Central cytologic, cytogenetic and histologic review was performed. Mutational analysis of TP53 was done by next generation sequencing (NGS). For generation of PCR amplicon libraries TP53 primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) study network. Amplicon sequencing of TP53 was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden. Results: A total of 91 patients were enrolled into the trial. Of those, 71 patients (male, n=13) were analyzed for TP53 mutations. Median age was 71 years (range 40-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). There was no difference between the TP53 mutated (TP53mut) versus TP53 wildtype patients (TP53WT) with regard to age, IPSS risk, hemoglobin levels, absolute neutrophil counts and platelet counts at baseline. Transfusion independence was achieved in a significantly lower proportion of patients in the TP53mut group versus TP53WT group (43% vs. 62%, p=0.036). Moreover, median survival was significantly shorter in the TP53mut group as compared to TP53WT group (533 days vs. not reached, p=0.0002). No difference was seen with regard to cytologic and cytogenetic response. Data on evolution into AML are currently being collected. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4-17 months on Len treatment (27% and 51%, respectively), although one of the patients achieved a complete cytogenetic response during Len treatment. Conclusions: Using the NGS technique on a routine basis, we achieved high quality runs with a high mean coverage of analyzed exons of TP53. Presence of TP53 mutations adversely affected response to Len with regard to transfusion independence. Moreover, TP53mut patients had a shorter overall survival as compared to TP53WT patients underlining the prognostic relevance of TP53 mutations in this patient cohort. Therefore, mutation analysis of TP53 might guide treatment decisions in the future, e.g. consideration of combination regimens. Since the TP53 clone obviously prevails during Len treatment, careful monitoring for signs of transformation should be performed. Disclosures Platzbecker: Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.1920.1920
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 6
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    Gene Expression of the Erythroid Regulator Erythroferrone (ERFE) is Highly Deregulated in CD71+ Erythroprogenitor Cells of Patients with Myelodysplastic Syndromes and Demonstrates Prognostic Relevance
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 4620-4620
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 4620-4620
    Abstract: Introduction Myelodysplastic Syndromes (MDS) are clonal hematologic diseases that are characterized by inefficient hematopoiesis, severe anemia and resulting deregulated iron homeostasis. Apart from supportive therapy with red blood cell transfusions some MDS patients with anemia respond to treatment with erythropoiesis stimulating agents such as Erythropoietin (EPO). However, the majority of these patients also become refractory to EPO treatment during the course of disease, suggesting a dysfunctional regulation of erythropoiesis downstream of EPO signaling in MDS. Most recently, a crucial erythroid regulator of iron metabolism named Erythroferrone (ERFE) was newly discovered, which is selectively produced by bone marrow (BM) erythroprogenitor cells during hematopoietic stress and EPO stimulation (Kautz et al. ASH plenary session 2013 and Kautz et al., Nature Genetics 2014). Aberrant expression of ERFE has been shown to directly result in critical impairment of erythropoiesis. We therefore sought to examine the role of ERFEexpression in CD71+ erythroprogenitor cells derived from patients with MDS and secondary acute myeloid leukemia (sAML). Methods CD71+ erythroprogenitor cells were immunomagnetically isolated from ficollized mononuclear BM cells of patients suffering from MDS (n=86, IPSS-low/int-1-risk n=69, IPSS-int-2/high-risk n=17), sAML (n=18) and age-matched healthy donors (n=17). In addition to CD71+ cells, CD34+, CD61+, CD15+ selected BM as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old donors. After total RNA was extracted using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR and normalized to corresponding GPIhousekeeping gene expression levels. Patient follow up (FU) data was available for n=55 MDS and n=14 sAML samples. Results Analysis of ERFE expression in CD34+, CD15+, CD61+ and CD71+ BM as well as CD3+ and unselected mononuclear PB cells from MDS patients and healthy donors revealed almost exclusive expression of ERFE in CD71+ erythroprogenitor cells irrespective of disease state. Our analysis of ERFE expression profiles in this specific cell subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.1, p 〈 0.0001), IPSS-int-2/high-risk (FC=4.6, p=0.0003) and sAML (FC=6.5, p 〈 0.0001) relative to age-matched healthy controls. Despite this marked profile of aberrantly regulated ERFE we identified a distinct fraction of patients with expression levels similar or even lower than those measured in healthy donors in 20% (11/55) and 36% (5/14) of analyzed MDS and sAML cases with FU. Univariate analysis revealed that low abundance of CD71+ ERFE transcripts was significantly associated with inferior overall survival (OS) in MDS patients (median survival 1.7 years vs. not reached, p=0.0066) and also sAML (median survival 0.1 vs. 0.8 years, p=0.031). Conclusion The recent identification of the novel key regulatory gene ERFE in mouse models has greatly improved the understanding of the dynamic regulation of erythropoiesis. Our observation of almost exclusive ERFE expression in human BM erythroprogenitor cells further underlines its important role in human erythropoietic regulation both in healthy and myelodysplastic hematopoiesis. Moreover, strong upregulation of erythropoiesis stimulating ERFE in a large proportion of MDS patients usually suffering from anemia likely indicates its involvement in perturbed mechanisms of feedback signaling in MDS erythropoiesis. Pending integration with further clinical data, the current observation of significantly inferior survival probability for MDS and sAML patients with low ERFE expression levels indicates the potentially important biologic and clinical relevance of this novel regulatory gene in the pathogenesis of MDS. Consequently, aberrant levels of the erythroid hormone ERFEin MDS erythroprogenitor cells might provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS. Disclosures Nolte: Celgene Corp., Novartis Pharma: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.4620.4620
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 7
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    Genome Wide DNA Methylation Analysis of Patients with Myelodysplastic Syndrome and Isolated Deletion (5q) Reveals Characteristic Methylation Profiles in Low and Intermediate-1 Risk Groups
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 3801-3801
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    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 3801-3801
    Abstract: Abstract 3801 Background Aberrant DNA methylation at promoter CpG islands is recognized as one of the hallmarks driving the pathogenesis of myeloid malignancies, especially myelodysplastic syndromes (MDS). Shen et al. recently characterized an algorithm of 10 aberrantly methylated gene loci which was predictive for overall survival and progression-free survival in a large cohort of MDS patients. A particular cytogenetic subgroup of MDS patients with a deletion on the long arm of chromosome 5 (5q-syndrome) has been shown to benefit from treatment with lenalidomide. However, the exact underlying molecular mechanism of MDS with isolated deletion (5q) is still not understood. To further elucidate on the role of deregulated DNA methylation we analyzed DNA-methylation profiles of bone marrow cells from patients with MDS with an isolated deletion (5q). Methods All patients were diagnosed and treated within a German multicenter trial investigating the safety of Lenalidomide in patients with low risk myelodysplastic syndromes and an isolated deletion (5q) after informed consent and according to the declaration of Helsinki. Bone marrow cells of 47 MDS patients with deletion (5q) at initial diagnosis were analyzed (median age 70 years, range 41 – 88 years, IPSS score: low n= 22; intermediate-1 n = 25). DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 15% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Gene ontology analysis was performed using GATHER (http://http://gather.genome.duke.edu/). Results Using a q-value of ≤ 0.05 for the beta-value of the array and excluding gender-specific chromosomal CpGs, 473,929 CpGs were evaluable for analysis. Gene Ontology analysis using the GATHER Tool showed a significant enrichment of genes mapped to 5q31 (p 〈 0.0001). Highly significant differential methylation profiles between MDS patients with isolated (5q) were found between patients with low and intermediate-1 IPSS score. CpGs differentially hypermethylated in intermediate-1 risk versus lows risk patients affected the coding regions of interesting candidate genes such as platelet-derived growth factor receptor, beta polypeptide (PDGFRB), clathrin interactor 1 (CLINT1), both located at 5q33 and suspected to be involved in the pathogenesis of 5q deleted MDS. Furthermore, transcriptional regulators such as proline, glutamate and leucine rich protein 1 (PELP1), v-myb myeloblastosis viral oncogene homolog (avian) (MYB), genes known to be involved in cancer like trichorhinophalangeal 1(TRPS1), and tumor suppressors like forkhead box P1 (FOXP1) and genes thought to be involved in the pathogenesis of MDS like Minichromosome maintenance protein2(MCM2) did show differentially DNA-methylation according to our selection criteria. Conclusions We present a comprehensive genome wide methylation analysis of MDS patients with an isolated deletion (5q) with low and intermediate-1 risk according to IPSS. Thereby we detected sets of significantly differentially methylated CpGs between both risk groups. Correlation of these data to clinical parameters might help to further elucidate the contribution of aberrant methylation to the phenotype of MDS with isolated deletion (5q) and could possibly help establishing novel prognostic markers based on differential methylation. Moreover, unraveling the role of aberrant methylation patterns might result in new therapeutic treatment approaches at least in a subset of patients. Disclosures: Nowak: Celgene: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ottmann:Celgene: Clinical trial participation Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Schlenk:Celgene: Research Funding. Ganser:Celgene: Research Funding. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hofmann:Celgene: Honoraria, Research Funding. Nolte:Celgene: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.3801.3801
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 8
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    Next Generation Sequencing-Based Molecular Dissection Of Lineage-Specific Mutational Hierarchies In Oligoclonal Primary and Xenografted Myelodysplasia
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 519-519
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 519-519
    Abstract: The underlying molecular defects in myelodysplastic syndromes (MDS), which are a heterogeneous group of malignant clonal hematologic disorders, are not well understood. Recently, next generation sequencing (NGS) based whole genome and exome sequencing highlighted the oligoclonal nature of persistent MDS clones that are present already at early disease stages. The reconstruction of mutational hierarchies in MDS clones and distinction of primary founder from subsequently acquired lesions has yet to be thoroughly interrogated and is likely to aid dissecting the molecular pathogenesis of MDS. Methods An amplicon-based NGS assay using the Roche 454 GS Junior system was established within the IRON-II framework study in order to screen for 17 commonly mutated genes in MDS. Genomic DNA from purified mononuclear bone marrow (BM) cells of 23 MDS IPSS low/int1 risk subjects was screened for somatic mutations. Called variants were compared to dbSNP and COSMIC database entries to rule out germline polymorphisms. In addition, copy number variation analysis was performed by Affymetrix SNP 6.0 array profiling. Custom pyrosequencing assays and interphase-FISH were applied for sensitive quantification of lesion burdens in FACS-sorted myeloid, erythroid, lymphoid and stem/progenitor cells. These were isolated from patients’ primary BM as well as their long-term engrafted human xenotransplants using our recently established MDS xenograft model. Results In this work, we identified 12 oligoclonal BM samples with ≥2 molecular lesions. Of note, varying frequencies of individual mutations between different sorted cell subsets from primary or human xenografted BM support the notion that distinct MDS (sub-)clones from these subjects contributed to hematopoiesis simultaneously and lead to differential engraftment between xenografts. Comparison of variable subset-specific mutation burdens allowed deciphering the individual hierarchical architecture of the mutational landscape from 9 individuals. ASXL1, SF3B1 and SRSF2 were detected as a primary lesion for 2 patients each. In contrast, large-scale genomic alterations such as del(5q), del(RUNX1) or trisomy 8 occurred as late-end lesion or even defined distinct clones which coexist with others harboring different mutations as detected for 2 subjects. Surprisingly, CD19+ and CD3+ lymphocytes from primary and/or xenografted BM displayed significant mutational burden of at least 1 mutation in 50% of the MDS cohort (5/10). Moreover, mutations were detected simultaneously in lymphocytes (hCD19+) as well as myeloid (hCD33+) and erythroid (hCD235a+) cells from three xenografted samples indicating a potent multilineage engraftment capability of MDS hematopoietic stem cells. Interestingly, one individual presented with high RUNX1 mutational frequency in the primary early progenitor fraction (CD34+CD38+), which was absent in the stem-cell enriched fraction (CD34+CD38-), whereas TET2, ZRSR2 and ASXL1 mutations were detected in both fractions and their xenografts. Intriguingly, only xenotransplantation of primary CD34+38- BM cells lead to long-term engraftment of RUNX1 wild type human BM cells in mice, while CD34+CD38+ BM cells gave rise to short term engraftment of RUNX1 mutated human BM cells indicating that mutated RUNX1might originate in a more committed progenitor fraction with limited self-renewal potential. Conclusion Molecular characterization of oligoclonal mutation patterns in primary and xenograft BM allowed the establishment of individual mutational hierarchies and indicates a relatively random order in the mutational evolution of MDS clones, although spliceosome mutations appear as rather early events. Furthermore, our analysis revealed engraftment of independent MDS clones in different mice xenografted with the same subject material, which opens the door to the in vivo study of isolated clones with respect to their pathomechanisms and response to treatment. Our data also suggests that the occurrence of large-scale genomic aberrations is frequently preceded by small-scale gene mutations, emphasizing their potential role in disease diagnosis and risk stratification. Finally, detection of MDS specific mutations in the lymphocytic compartment might be involved in facilitating impaired immune functionality and needs to be investigated prospectively. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Staller:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.519.519
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 9
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    Development of a DNA-Based Targeted Assay Suitable for Accurate Quantification of Chromosomal Deletions in Myelodysplastic Syndromes with Deletion (5q) and Other Clonal Diseases
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 1925-1925
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    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1925-1925
    Abstract: Introduction The acquisition of large-scale chromosomal lesions is a frequent event in malignant disorders. One example is the recurrent deletion of chromosome 5q (del(5q)) in myelodysplastic syndromes (MDS). The detection and monitoring of such deletions are important elements in routine clinical diagnostics and cancer genomic studies. Currently established methods for their assessment are metaphase cytogenetics (MC), fluorescence in situ hybridization (FISH) and micro-array based techniques. However, each of these methods harbours specific disadvantages as they depend on (viable) cells, are expensive, labour-intensive or only semi-quantitative. One possible approach to interrogate chromosomal deletions constitutes the assessment of allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Therefore, we aimed to establish a robust, quick and inexpensive PCR assay that measures allelic imbalance at such STR loci in order to reliably estimate frequencies of cells carrying del(5q) from only minute amounts of DNA. Methods Genomic DNA (gDNA) was isolated from bone marrow (BM) or blood cells of MDS and acute myeloid leukemia (AML) patients with cytogenetically confirmed del(5q). Based on NCBI UniSTS database, we designed 12 fluorochrome-labelled PCR amplicons with size ranges of 100-400 bp that surround STR loci between chromosomal bands 5q21 and 5q31. Using only 10 ng gDNA, all 12 PCR amplicons were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis on an ABI 3130 Genetic Analyzer. Allele size quantification of informative heterozygous loci was performed using ABI Genemapper software. Furthermore, size calculations of individual alleles were corrected for PCR-stutter, which was estimated from corresponding loci in homozygous samples. Finally, using mesenchymal stromal cells as a germline control, the degree of skewing in the allelic ratios of all informative STR markers in the tumor sample relative to the corresponding allelic ratios in the control was averaged and subsequently translated into fractions of cells carrying the del(5q) lesion. Results Application of our novel assay for quantification of del(5q) burden in n=559 samples from n=67 patients revealed a high frequency of informative markers with an average of 7 heterozygous STR loci per patient. The data shows a strong inter-marker concordance with a standard deviation (SD) of 2.3% for del(5q) cell frequencies. Moreover, duplicate analysis of 328 samples revealed an average SD of 0.86%. Most importantly, paired analysis of the proportion of del(5q) cells estimated using interphase-FISH and our PCR assay was carried out for n=9 samples and resulted in strong correlation with r²=0.93. A serial dilution series with deleted and non-deleted gDNA also revealed highly concordant results with r²=0.96. When comparing matched germline and tumor STR profiles, no case of microsatellite instability was detectable in our MDS/AML cohort thus highlighting the suitability of STR based lesion quantification in this disease entity. Furthermore, we could use our large dataset to calculate amplification efficiencies for each locus in order to predict “surrogate” germline profiles eventually allowing us to calculate del(5q) frequencies in tumor samples that lack germline controls. Finally, our assay reliably confirmed molecular remission in BM from del(5q) MDS patients after Lenalidomide treatment, in agreement with concomitant MC analyses. Conclusion Using a multiplexed PCR assay for measurement of STRs in deleted chromosomal regions we present a highly adaptable tool for precise quantification of large scale lesions. We show a very good correlation with established methods exemplarily for del(5q) lesions. Even without available germline control our assay provides robust results. Requiring only minute amounts of gDNA, this assay is ideally suitable for copy number quantification in samples for which only residual archival gDNA and no cells for FISH analysis are available. Due to the small amplicon sizes it should also be useful for investigation of fragmented gDNA from formalin-fixed archival specimen. In summary, our newly developed assay offers a mean to obtain quantitative data for basically any large scale chromosomal deletion which contains STRs and should be easily applicable to other clonal diseases. Disclosures Nolte: Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.1925.1925
    RVK:
    XA 33000
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    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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    Genome Wide DNA Methylation Analysis Identifies a Characteristic Methylation Profile for Patients with Acute Promyelocytic Leukemia and FLT3-Internal Tandem Duplication
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 3521-3521
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 3521-3521
    Abstract: Abstract 3521 Background Acute promyelocytic leukemia (APL) accounts for approximately 5% of all acute myeloid leukemias (AML). The characteristic molecular feature of APL is a fusion product named PML-RARA which acts as transcriptional repressor that affects gene expression patterns involved in differentiation, apoptosis, and self-renewal. The internal tandem duplication of the Fms-related Tyrosine-like Kinase 3 (FLT3-ITD) confers a poor prognosis in non-APL AML, however its effect in APL is still under discussion as several investigators found no prognostic influence for FLT3-ITD in APL. Aberrant DNA-promotor-methylation of tumor suppressor genes contributes significantly to leukemogenesis and oncogenic transformation. Deneberg et al. recently identified characteristic methylation profiles for cytogenetically normal AML, however no specific methylation profile was associated with FLT3-ITD in a study that excluded APL. To further elucidate the influence of aberrant methylation in FLT3-positive APL we carried out a genome wide DNA methylation analysis on APL samples with and without FLT3-ITD. Methods In total, genomic DNA from blasts of 54 APL patients at initial diagnosis (bone marrow n=32, peripheral blood n=22) were analyzed (median age 46 years, gender: 35 female, 19 male, blast count median 80%). The molecular analysis was carried out with written informed consent, with permission of the institutional review board and in accordance with the declaration of Helsinki. DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 25% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Pyrosequencing was performed to validate methylation changes as detected by the array-based analysis. Results The methylation pattern of FLT3-ITD-positive APL (n=18) patients was analyzed and compared to patients without FLT3-ITD (n=32) or D835 Mutation (n=4). We identified 133 CpGs that were significantly differentially methylated in FLT3-ITD-positive APL as compared to FLT3-ITD-negative APL. The most significant differential methylation was observed for 5 CpGs showing a strong hypomethylation of the chemokine (C-C motif) receptor 6 (CCR6) in FLT3-ITD-APL as compared to FLT3-negative APL (q-value 〈 6.9 *10−13). Other interesting target genes showing pronounced hypomethylation in FLT3-ITD positive APL samples belonged to the family of phosphatases such as the dual specificity phosphatase 5 (DUSP5), protein tyrosine phosphatase, receptor type, N polypeptide 2 (PTPRN2) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). The most prominent hypermethylation in FLT3-ITD APL was observed in CpGs within the coding region of suppressor of cytokine signaling 2 (SOCS2) and significantly discriminated between FLT3-ITD and FLT3-negative APL (q-value 〈 10−5). The results of the genome-wide analysis obtained with the Illumina 450K BeadChip were validated for 4 CPGs in 10 samples via pyrosequencing and showed a robust Pearson correlation of 0.92 suggesting a good and reliable performance of the Illumina 450 K Bead Chip Assay. Conclusions The current study represents a comprehensive genome wide methylation analysis of a clinically well-defined cohort of APL patients. We here demonstrate for the first time that in contrast to cytogenetically normal AML, APL patients with FLT3-ITDs display a highly specific and disease defining DNA methylation profile. Thereby key regulators of cellular growth signaling such as SOC2, PTPRN2 and DUSP5 are significantly differentially methylated in dependency of FLT3-ITD status. This suggests that a cooperative effect between PML-RARA and FLT3-ITD is mediated by dysregulation of DNA methylation. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.3521.3521
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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    detail.hit.zdb_id: 80069-7
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