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Huang D.,Capital Medical University | Huang D.,Cleveland Clinic | Nagata Y.,Kyoto University | Grossmann V.,Munich Leukemia Laboratory MLL | And 36 more authors.
Haematologica | Year: 2015

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms. © 2015 Ferrata Storti Foundation.


Piazza R.,University of Milan Bicocca | Valletta S.,University of Milan Bicocca | Winkelmann N.,Salisbury District Hospital | Winkelmann N.,Universitatsklinikum Jena | And 32 more authors.
Nature Genetics | Year: 2013

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases. © 2013 Nature America, Inc. All rights reserved.


Videm P.,Albert Ludwigs University of Freiburg | Rose D.,Albert Ludwigs University of Freiburg | Rose D.,Munich Leukemia Laboratory MLL | Costa F.,Albert Ludwigs University of Freiburg | And 2 more authors.
Lecture Notes in Informatics (LNI), Proceedings - Series of the Gesellschaft fur Informatik (GI) | Year: 2014

Sequence and secondary structure analysis can be used to assign putative functions to non-coding RNAs. However sequence information is changed by post-transcriptional modifications and secondary structure is only a proxy for the true 3D conformation of the RNA polymer. In order to tackle these issues we can extract a different type of description using the pattern of processing that can be observed through the traces left in small RNA-seq reads data. To obtain an efficient and scalable procedure, we propose to encode expression profiles in discrete structures, and process them using fast graph-kernel techniques. We present BlockClust for both clustering and classification of small non-coding RNA transcripts with similar processing patterns. We show how the proposed approach is scalable, accurate and robust across different organisms, tissues and cell lines. BlockClust was successfully applied on a comprehensive set of eukaryotic data. It is the first tool for eukaryotic non-coding RNA analysis available on the galaxy framework.


Videm P.,Albert Ludwigs University of Freiburg | Rose D.,Albert Ludwigs University of Freiburg | Rose D.,Munich Leukemia Laboratory MLL | Costa F.,Albert Ludwigs University of Freiburg | And 2 more authors.
Bioinformatics | Year: 2014

Non-coding RNAs (ncRNAs) play a vital role in many cellular processes such as RNA splicing, translation, gene regulation. However the vast majority of ncRNAs still have no functional annotation. One prominent approach for putative function assignment is clustering of transcripts according to sequence and secondary structure. However sequence information is changed by post-transcriptional modifications, and secondary structure is only a proxy for the true 3D conformation of the RNA polymer. A different type of information that does not suffer from these issues and that can be used for the detection of RNA classes, is the pattern of processing and its traces in small RNA-seq reads data. Here we introduce BlockClust, an efficient approach to detect transcripts with similar processing patterns. We propose a novel way to encode expression profiles in compact discrete structures, which can then be processed using fast graph-kernel techniques. We perform both unsupervised clustering and develop family specific discriminative models; finally we show how the proposed approach is scalable, accurate and robust across different organisms, tissues and cell lines. © 2014 The Author. Published by Oxford University Press. All rights reserved.


Medyouf H.,German Cancer Research Center | Mossner M.,University of Mannheim | Jann J.-C.,University of Mannheim | Nolte F.,University of Mannheim | And 29 more authors.
Cell Stem Cell | Year: 2014

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. © 2014 Elsevier Inc.


Roug A.S.,Aarhus University Hospital | Nyvold C.G.,Aarhus University Hospital | Juhl-Christensen C.,Aarhus University Hospital | Christensen M.,Aarhus University Hospital | And 2 more authors.
European Journal of Haematology | Year: 2011

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell. © 2011 John Wiley & Sons A/S.


Lengfelder E.,University of Mannheim | Lengfelder E.,Universitatsmedizin Mannheim | Hanfstein B.,University of Mannheim | Haferlach C.,Munich Leukemia Laboratory MLL | And 21 more authors.
Annals of Hematology | Year: 2013

Despite improvement of prognosis, older age remains a negative prognostic factor in acute promyelocytic leukemia (APL). Reports on disease characteristics and outcome of older patients are conflicting. We therefore analyzed 91 newly diagnosed APL patients aged 60 years or older (30 % of 305 adults with APL) registered by the German AML Cooperative Group (AMLCG) since 1994; 68 patients (75 %) were treated in studies, 23 (25 %) were non-eligible, and 31 % had high-risk APL. Fifty-six patients received induction therapy with all-trans retinoic acid and TAD (6-thioguanine, cytarabine, daunorubicin), and consolidation and maintenance therapy. Treatment intensification with a second induction cycle (high dose cytarabine, mitoxantrone; HAM) was optional (n = 14). Twelve patients were randomized to another therapy not considered in this report. The early death rate was 48 % in non-eligible and 19 % in study patients. With the AMLCG regimen, 7-year overall, event-free and relapse-free survival (RFS) and cumulative incidence of relapse were 45 %, 40 %, 48 %, and 24 %, respectively. In patients treated with TAD-HAM induction, 7-year RFS was superior (83 %; p = 0.006) compared to TAD only, and no relapse was observed. In our registered elderly patients, we see a high rate of non-eligibility for treatment in studies and of high-risk APL. In patients who can undergo a curative approach, intensified chemotherapy is highly effective, but is restricted to a selection of patients. Therefore, new less toxic treatment approaches with broader applicability are needed. Elderly patients might be a particular target group for concepts with arsenic trioxide. © 2012 The Author(s).


PubMed | Munich Leukemia Laboratory MLL, Kyoto University, Cedars Sinai Medical Center, Cleveland Clinic and 5 more.
Type: Journal Article | Journal: Haematologica | Year: 2015

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.


Bacher U.,University of Hamburg | Haferlach T.,Munich Leukemia Laboratory MLL | Schnittger S.,Munich Leukemia Laboratory MLL | Kreipe H.,Hannover Medical School | Kroger N.,University of Hamburg
British Journal of Haematology | Year: 2011

The clinical, morphological, and genetic heterogeneity of chronic myelomonocytic leukaemia (CMML), has made it difficult to clearly assign this entity to a distinct haematological category. In 2001, the World Health Organization transferred CMML to a new category of mixed myeloproliferative/myelodysplastic disorders, which was maintained in the last revision in 2008. Considering the rare occurrence of CMML, most pharmacotherapeutic and transplant studies combined CMML with myelodysplastic syndrome cases, but some clinical trials specifically investigated the use of demethylating agents in CMML and demonstrated stabilization of the haematological situation or even complete remission in subsets of patients. Information on the significance of other drugs is very limited. Allogeneic haematopoietic stem cell transplantation (HSCT) remains the only curative option for patients with CMML. Molecular studies revealed various novel genetic alterations in CMML - notably of the JAK2, TET2, CBL, IDH, or RUNX1 and RAS genes. This review summarizes the current status of pharmacotherapy and transplantation in CMML and outlines recent results of molecular research for diagnosis of this heterogeneous entity. © 2011 Blackwell Publishing Ltd.

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