216 results on '"Schwabe, A."'
Search Results
2. Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition
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Irving, Julie, Matheson, Elizabeth, Minto, Lynne, Blair, Helen, Case, Marian, Halsey, Christina, Swidenbank, Isabella, Ponthan, Frida, Kirschner-Schwabe, Renate, Groeneveld-Krentz, Stefanie, Hof, Jana, Allan, James, Harrison, Christine, Vormoor, Josef, von Stackelberg, Arend, and Eckert, Cornelia
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- 2014
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3. Subclonal NT5C2 mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia
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Arend von Stackelberg, Hossein Khiabanian, Jui Wan Loh, Stefanie Groeneveld-Krentz, Adolfo A. Ferrando, Malwine J. Barz, Annabell Szymansky, Jana Hof, Cornelia Eckert, Kathy Astrahantseff, and Renate Kirschner-Schwabe
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Male ,Oncology ,medicine.medical_specialty ,Adolescent ,DNA Mutational Analysis ,Immunology ,Purine analogue ,medicine.disease_cause ,Biochemistry ,law.invention ,Young Adult ,Gene Frequency ,Recurrence ,law ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Humans ,Young adult ,Allele ,Child ,5'-Nucleotidase ,Allele frequency ,Alleles ,Polymerase chain reaction ,Mutation ,business.industry ,Hazard ratio ,Wild type ,Infant ,Cell Biology ,Hematology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,Prognosis ,Child, Preschool ,Female ,business ,Biomarkers - Abstract
Activating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.
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- 2020
4. TP53 and KRAS Variants at Initial Diagnosis Identify an Ultra-High Risk Group of Pediatric T-Lymphoblastic Leukemia (T-ALL)
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Kempter, Tamara, primary, Kunz, Joachim B., additional, Richter-Pechanska, Paulina, additional, Tomska, Katarzyna, additional, Rausch, Tobias, additional, Erarslan-Uysal, Busra, additional, Eckert, Cornelia, additional, Zimmermann, Martin, additional, Stanulla, Martin, additional, Schrappe, Martin, additional, Cario, Gunnar, additional, Escherich, Gabriele, additional, Kirschner-Schwabe, Renate, additional, Muckenthaler, Martina U., additional, Korbel, Jan O., additional, and Kulozik, Andreas E., additional
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- 2021
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5. CD11b is a therapy resistance– and minimal residual disease–specific marker in precursor B-cell acute lymphoblastic leukemia
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Rhein, Peter, Mitlohner, Rita, Basso, Giuseppe, Gaipa, Giuseppe, Dworzak, Michael N., Kirschner-Schwabe, Renate, Hagemeier, Christian, Stanulla, Martin, Schrappe, Martin, Ludwig, Wolf-Dieter, Karawajew, Leonid, and Ratei, Richard
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- 2010
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6. The AF4·MLL fusion protein is capable of inducing ALL in mice without requirement of MLL·AF4
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Bursen, Adelheid, Schwabe, Karen, Rüster, Brigitte, Henschler, Reinhard, Ruthardt, Martin, Dingermann, Theo, and Marschalek, Rolf
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- 2010
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7. TP53 and KRAS Variants at Initial Diagnosis Identify an Ultra-High Risk Group of Pediatric T-Lymphoblastic Leukemia (T-ALL)
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Katarzyna Tomska, Martin Stanulla, Joachim B. Kunz, Andreas E. Kulozik, Renate Kirschner-Schwabe, Tamara Kempter, Cornelia Eckert, Martina U. Muckenthaler, Jan O. Korbel, Gunnar Cario, Gabriele Escherich, Martin Schrappe, Büşra Erarslan-Uysal, Martin Zimmermann, Tobias Rausch, and Paulina Richter-Pechanska
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Oncology ,medicine.medical_specialty ,business.industry ,Lymphoblastic Leukemia ,Immunology ,Cell Biology ,Hematology ,Ultra high risk ,medicine.disease_cause ,Biochemistry ,Internal medicine ,Medicine ,KRAS ,business - Abstract
Introduction Patients who suffer a relapse of pediatric T-cell acute lymphoblastic leukemia (T-ALL) face a dismal prognosis. Prognostic molecular biomarkers that reliably predict the risk of relapse at the time of first diagnosis are not available. Inactivating mutations in TP53 were previously detected in approximately 10% of relapsed patients (Hof et al. J Clin Oncol. 2011) and are invariably associated with fatal outcome (Richter-Pechanska et al. Blood Cancer J. 2017). Mutations in other genes were identified to be either specific for relapse (NT5C2 and CCDC88A) or to be associated with a poor prognosis in relapse (IL7R, KRAS, NRAS, USP7, CNOT3 and MSH6) (Meyer et al. Nat Genet. 2013; Richter-Pechanska et al. Blood Cancer J. 2017). We hypothesized that subclones bearing such mutations can give rise to relapse and analyzed these 9 genes at initial diagnosis of T-ALL with targeted ultra-deep sequencing. Methods Leukemia samples collected at initial diagnosis of 81 children with T-ALL who later relapsed were analyzed. As a control group, we selected 79 children with T-ALL who remained in first remission for at least three years and were matched with regard to treatment response, treatment, age and sex. Targeted deep sequencing was performed by using the Agilent Haloplex High Sensitivity kit with unique molecular identifiers for reliable detection of mutations with very low allele frequencies (average read depth: 1,012x). Results Overall, we detected 75 mutations among 7 targeted genes in 33 / 81 relapsing and 21 / 79 non-relapsing patients. The average allele frequency (AF) of the identified mutations was 25% (0.8% - 83%; SD ± 18%). More than half of the variants (43/75) showed AFs below 30% and were thus classified as subclonal. Interestingly, 7 pathogenic TP53 mutations (subclonal: n=5, clonal: n=2) with AFs of 4.4% - 49.4% were exclusively discovered in 6 patients who experienced a relapse. While 2 of these patients received an allogeneic stem cell transplantation in first remission because of poor treatment response, the remaining 4 patients were treated by chemotherapy in the high-risk (n=1) or medium-risk (n=3) arm. None of the 79 non-relapsing control patients carried TP53 mutations. Consistent with the hypothesis of clonal evolution as a mechanism of relapse in T-ALL, Sanger Sequencing of the relapse sample of one TP53-positive patient confirmed that the subclone harboring the TP53 mutation A159D at initial diagnosis (AF 5.4%) expanded to a major clone (AF 42%) in relapse. The presence of TP53 mutations in two further TP53-positive patients in at least one available post-remission sample is also compatible with clonal selection. However, in a fourth patient the low allele frequency of the TP53 mutation at relapse indicates that the TP53 subclone persisted but did not expand during the development of relapse. In addition to TP53, we identified pathogenic KRAS mutations to be significantly enriched in relapsing patients (9 / 81) compared to non-relapsing patients (2 / 79) at the time of initial diagnosis (chi-squared test, p= 0.032; Table 1). Conclusion Subclonal and clonal mutations in TP53 and KRAS at initial diagnosis were enriched in T-ALL patients who later relapsed and identified approximately 17% of patients suffering a relapse. We thus propose that (subclonal) mutations of TP53 and KRAS may define a subgroup of high-risk T-ALL patients already at the time of first diagnosis. The identification of such mutations may complement the current risk stratification which depends on treatment response and may determine a new molecularly defined subgroup of T-ALLs that may benefit from intensified treatment strategies. Figure 1 Figure 1. Disclosures Schrappe: SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; Servier: Honoraria, Other: research support; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Novartis: Honoraria, Other: research support. Cario: Novartis: Other: Lecture Fee. Muckenthaler: Silence Therapeutics: Research Funding. Kulozik: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria.
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- 2021
8. Subclonal NT5C2 mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia
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Barz, Malwine J., primary, Hof, Jana, primary, Groeneveld-Krentz, Stefanie, primary, Loh, Jui Wan, primary, Szymansky, Annabell, primary, Astrahantseff, Kathy, primary, von Stackelberg, Arend, primary, Khiabanian, Hossein, primary, Ferrando, Adolfo A., primary, Eckert, Cornelia, primary, and Kirschner-Schwabe, Renate, primary
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- 2020
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9. Thromboembolic events in children with acute lymphoblastic leukemia (BFM protocols): prednisone versus dexamethasone administration
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Nowak-Göttl, Ulrike, Ahlke, Elvira, Fleischhack, Gudrun, Schwabe, Dirk, Schobess, Rosmarie, Schumann, Christiane, and Junker, Ralf
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- 2003
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10. Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes
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Erarslan-Uysal, Büşra, primary, Kunz, Joachim B., additional, Rausch, Tobias, additional, Richter-Pechanska, Paulina, additional, Waszak, Sebastian, additional, Frismantas, Viktoras, additional, Bornhauser, Beat, additional, Zimmermann, Martin, additional, Stanulla, Martin, additional, Schrappe, Martin, additional, Cario, Gunnar, additional, Escherich, Gabriele, additional, Bakharevich, Kseniya, additional, Kirschner-Schwabe, Renate, additional, Eckert, Cornelia, additional, Loukanov, Tsvetomir, additional, Gorenflo, Matthias, additional, Muckenthaler, Martina U., additional, Bourquin, Jean-Pierre, additional, Korbel, Jan O., additional, and Kulozik, Andreas E., additional
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- 2018
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11. Correlations of Plasma Cytokine Levels and Anti-FVIII Antibodies during Immune Tolerance Induction
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Hartmann, Johannes, primary, Schmidt, Anja, additional, Beilfuss, Marko, additional, Stichel, Diana, additional, Heller, Christine, additional, Schwabe, Dirk, additional, Klingebiel, Thomas, additional, Ewing, Nadia P, additional, and Koenigs, Christoph, additional
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- 2018
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12. Longitudinal Multilevel Omic Analysis of Pediatric T-ALL Reveals Distinct Mechanisms for Disease Progression in Type 1 and in Type 2 Relapses
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Richter-Pechanska, Paulina, primary, Kunz, Joachim B., additional, Rausch, Tobias, additional, Erarslan-Uysal, Busra, additional, Bornhauser, Beat, additional, Frismantas, Viktoras, additional, Dobay, Maria Pamela, additional, Von Knebel Doeberitz, Caroline, additional, Zimmermann, Martin, additional, Fuhrmann, Stephan, additional, Stanulla, Martin, additional, Schrappe, Martin, additional, Cario, Gunnar, additional, Escherich, Gabriele, additional, Bakharevich, Kseniya, additional, Kirschner-Schwabe, Renate, additional, Eckert, Cornelia, additional, Pfister, Stefan, additional, Muckenthaler, Martina U., additional, Bourquin, Jean-Pierre, additional, Korbel, Jan O., additional, and Kulozik, Andreas E., additional
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- 2018
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13. Frequency and epitope specificity of anti-factor VIII C1 domain antibodies in acquired and congenital hemophilia A
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Pete Lollar, Aleksander Orlowski, John F. Healey, Diana Stichel, Christoph Königs, Ernest T. Parker, Manuela Krause, Dirk Schwabe, Marc Jacquemin, Joerg Kahle, and Andreas Tiede
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0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,medicine.drug_class ,Swine ,animal diseases ,Immunology ,Protein domain ,030204 cardiovascular system & hematology ,Monoclonal antibody ,Hemophilia A ,Biochemistry ,Group A ,Epitope ,Immunoglobulin G ,Thrombosis and Hemostasis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Protein Domains ,hemic and lymphatic diseases ,Medicine ,Animals ,Humans ,Factor VIII ,biology ,business.industry ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,Fusion protein ,Virology ,Molecular biology ,Antibodies, Neutralizing ,030104 developmental biology ,Epitope mapping ,biology.protein ,Antibody ,business ,Epitope Mapping - Abstract
Several studies showed that neutralizing anti-factor VIII (anti-fVIII) antibodies (inhibitors) in patients with acquired hemophilia A (AHA) and congenital hemophilia A (HA) are primarily directed to the A2 and C2 domains. In this study, the frequency and epitope specificity of anti-C1 antibodies were analyzed in acquired and congenital hemophilia inhibitor patients (n = 178). The domain specificity of antibodies was studied by homolog-scanning mutagenesis (HSM) with single human domain human/porcine fVIII proteins and antibody binding to human A2, C1, and C2 domains presented as human serum albumin (HSA) fusion proteins. The analysis with HSA-fVIII domain proteins confirmed the results of the HSM approach but resulted in higher detection levels. The higher detection levels with HSA-fVIII domain proteins are a result of antibody cross-reactivity with human and porcine fVIII leading to false-negative HSM results. Overall, A2-, C1-, and C2-specific antibodies were detected in 23%, 78%, and 68% of patients with AHA (n = 115) and in 52%, 57%, and 81% of HA inhibitor patients (n = 63). Competitive binding of the human monoclonal antibody (mAb) LE2E9 revealed overlapping epitopes with murine C1-specific group A mAbs including 2A9. Mutational analyses identified distinct crucial binding residues for LE2E9 (E2066) and 2A9 (F2068) that are also recognized by anti-C1 antibodies present in patients with hemophilia. A strong contribution of LE2E9- and 2A9-like antibodies was particularly observed in patients with AHA. Overall, our study demonstrates that the C1 domain, in addition to the A2 and C2 domains, contributes significantly to the humoral anti-fVIII immune response in acquired and congenital hemophilia inhibitor patients.
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- 2016
14. Longitudinal Multilevel Omic Analysis of Pediatric T-ALL Reveals Distinct Mechanisms for Disease Progression in Type 1 and in Type 2 Relapses
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Renate Kirschner-Schwabe, Beat Bornhauser, Jean-Pierre Bourquin, Paulina Richter-Pechanska, Joachim B. Kunz, Büşra Erarslan-Uysal, Martin Zimmermann, Viktoras Frismantas, Cornelia Eckert, Kseniya Bakharevich, Tobias Rausch, Martin Schrappe, Caroline Von Knebel Doeberitz, Korbel Jo, Martina U. Muckenthaler, Stephan Fuhrmann, Stefan M. Pfister, Martin Stanulla, Andreas E. Kulozik, G Cario, Gabriele Escherich, and Maria Pamela Dobay
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Oncology ,medicine.medical_specialty ,Mutation ,DNA repair ,Immunology ,Clone (cell biology) ,O-6-methylguanine-DNA methyltransferase ,Cell Biology ,Hematology ,Disease ,Biology ,medicine.disease_cause ,Biochemistry ,Somatic evolution in cancer ,Internal medicine ,medicine ,Epigenetics ,Gene - Abstract
We aimed at understanding the relapse-driving processes in pediatric T-ALL and performed an integrated longitudinal multi-level omics analysis of 13 T-ALL patients at initial diagnosis (INI) and relapse (REL). We compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression, methylation levels and chromatin accessibility by ATAC-Seq. Aberrant expression of T-ALL transcription factors (TAL1, TAL2,LMO2, TLX1, TLX3, NKX2.4 and NKX2.5) was preserved from initial presentation to relapse in all patients. These leukemia-driving events defined the expression patterns, methylation profiles and the chromatin accessibility landscapes. A global differential analysis of the RNA-Seq data (DESeq2, padj We then focused our analysis on the 2 types of relapse in pediatric T-ALL, which we have previously defined on the basis of subclonal mutation profiles (Kunz et al., 2015). These types of relapse are characterized by either clonal evolution of cells derived from the major clone at initial presentation (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that is distinct from the predominant initial clone (type 2). When considering type 1 and type 2 relapses separately we identified a strong trend for type 2 relapses to acquire more mutations (p=0.0879, ttest) than type 1 relapses. Further to the known activating mutations in NT5C2 acquired at relapse by 8/13 patients no other mutations or CNAs were recurrently acquired in the relapses of this group of patients. However, mutations in proto-oncogenes or genes involved in DNA surveillance were acquired by 7/8 type 2 relapse patients in our series. Changes of CNAs also occurred more frequently in type 2 than in type 1 relapses (pval= 0.0267, ttest). This increased complexity on the genetic level was also apparent on the epigenetic level, with an increase of changes in the methylation pattern (mean difference in β value between INI and REL: type 1 - 0.00034; type2 - 0.002 (pval< 0.0001, chi2)), chromatin accessibility (number of differentially accessible ATAC-peaks: type 1 - 4 (0.006%) ; type 2 - 1018 (1.3%); (pval< 0.0001, chi2)) and on the expression level (number of differentially expressed genes: type 1 - 11; type 2 - 111, pval When considering differences between leukemias at the time of initial diagnosis, which later develop either type 1 or type 2 relapses we found 1.016 genes to be differentially expressed (524: up; 492: down in type 1; DE-Seq2: padj In sum, the multilevel omic comparison of pediatric T-ALL that develop either a type 1 or a type 2 relapse show remarkably more complex changes of the genetic and epigenetic profiles during the transition from initial to relapsing disease. Notably, pediatric T-ALLs, who later develop a type 1 relapse display an epigenetic and transcriptomic landscape predicting an upregulation of DNA repair functions, which we suggest to potentially play a role in developing resistance to DNA damaging agents in this type of relapse. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.
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- 2018
15. Pediatric T-ALLs Developing into a Type 2 Relapse Originate from Cells That Carry the Potential of Variable Maturation into Subclones with Distinct Chromatin Landscapes
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Tsvetomir Loukanov, Beat Bornhauser, Jan O. Korbel, Andreas E. Kulozik, Cornelia Eckert, Martina U. Muckenthaler, Matthias Gorenflo, Martin Zimmermann, Renate Kirschner-Schwabe, Joachim B. Kunz, Martin Schrappe, Gunnar Cario, Büşra Erarslan-Uysal, Tobias Rausch, Gabriele Escherich, Martin Stanulla, Kseniya Bakharevich, Viktoras Frismantas, Sebastian M. Waszak, Jean-Pierre Bourquin, and Paulina Richter-Pechanska
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Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Chromatin ,Fusion gene ,Leukemia ,Early maturation ,medicine ,Progenitor cell ,CD8 ,Progenitor ,Epigenomics - Abstract
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is classified according to surface marker expression. In order to reveal the cells of origin in pediatric T-ALL and to understand mechanisms of relapse we used ATAC-Seq (Assay for Transposase-Accessible Chromatin Sequencing) to compare chromatin accessibility landscapes of healthy T-cell precursors to those of T-ALL cells obtained at initial diagnosis (INI) and relapse (REL). We have FACS sorted 7 differentiation stages of normal T-cell precursors contained in the thymus of infants undergoing cardiac surgery (DN2, DN3, ISP, DPCD3-, DPCD3+, CD4+ and CD8+) and subjected these to ATAC-Seq. Unsupervised learning by principal component analysis (PCA) clustered sorted populations according to the maturation stage, demonstrating that regulatory chromatin signatures of thymocytes are highly stage-specific and re-shaped during T-cell differentiation. We next compared normal T-cell precursors at different stages of maturation to pediatric T-ALLs and found fundamental differences with 30% of open chromatin regions to be more and 28% being less accessible in T-ALL (DESeq2, padj We then subjected the ATAC-seq data of all matched leukemia samples obtained at initial disease and at relapse to PCA. INI and REL samples derived from the same patient always clustered in close proximity and were separated according to the T-ALL driving fusion genes. A global analysis of differential accessibility revealed only 0.26% of ATAC-regions to be less- or more-accessible at relapse when compared to the matched initial samples (DESeq2, padj Moreover, we trained the deconvolution algorithm CIBERSORT to recognize particular T-cell differentiation stages using ATAC-profiles of the 7 FACS-sorted healthy T-cell populations. We used regulatory chromatin landscape of non-sorted (total) thymus to assess the accuracy of deconvolution. Comparison of predicted fractions in total thymus to FACS measurements revealed highly accurate identification of the maturation stages (r2 = 0.95). CIBERSORT analysis confirmed that the profiles were largely preserved between INI and REL of each sample pair. Notably, however, while in T-ALLs that later developed into a type 1 relapse only one type of early T-cell progenitor dominated the deconvolution profile, T-ALLs that developed into a type 2 relapse showed heterogeneous profiles with contributions of progenitors at different maturation stages. In sum, these epigenomic analyses revealed that the chromatin landscape of normal T-cell precursors evolves in the course of thymic maturation and that the early maturation stages are the likely origin of T-ALL cells. Remarkably, pediatric T-ALLs that later develop a type 2 relapse consist of subclones with a variable profile of chromatin accessibility that define different stages of maturation. These data indicate that T-ALLs with the propensity to develop a type 2 relapse differ from type 1 in that they originate from early precursors that carry the potential of further development into different stages of maturation before the leukemia becomes apparent with a highly subclonal pattern. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.
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- 2018
16. Correlations of Plasma Cytokine Levels and Anti-FVIII Antibodies during Immune Tolerance Induction
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Diana Stichel, Johannes Hartmann, Nadia P. Ewing, Marko Beilfuss, Anja Schmidt, Christine Heller, Dirk Schwabe, Christoph Koenigs, and Thomas Klingebiel
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medicine.medical_specialty ,biology ,business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Gastroenterology ,Immunoglobulin G ,Immune tolerance ,Interleukin 10 ,Titer ,Cytokine ,Aldesleukin ,Internal medicine ,biology.protein ,Medicine ,Interleukin 17 ,Antibody ,business - Abstract
Introduction The development of neutralizing anti-FVIII antibodies (inhibitors) with reduced or absent activity of substituted factor VIII (FVIII) remains the most serious complication of hemophilia A therapy (Kempton, 2014). Frequent and high doses of FVIII with or without bypassing products can reestablish immune tolerance in 60-70% of patients. Polymorphism in immune response genes including IL-10 and TNFa were identified as risk factors for inhibitor development (Astermark, 2006). Cross-sectional studies showed different cytokine profiles in patients with hemophilia, especially in those with history of an inhibitor (Oliveira, 2013). In this study cytokine profiles were monitored longitudinally during immune tolerance induction (ITI). Methods 107 plasma samples from 18 patients were collected during the RES.I.S.T Experienced and Naive trial, which included patients with a poor prognosis for ITI success (Gringeri, 2007). We quantified 14 cytokines in each sample by using a Human High Sensitivity T-Cell Discovery Array 14-Plex (Eve Technologies Corp, Calgary, AB, Canada). ELISA based FVIII antibody assays were used for anti-FVIII IgG detection. FVIII inhibitor titers (Bethesda assay, BU) were measured and available for the analysis. The cut-off for a positive inhibitor was >0,6 BU mL-1. Bethesda titers (BUpos) between 0,6 - Results Plasma levels of TNFa (P=0,014) and IL-8 (P=0,048) were positively correlated with FVIII inhibitor titers. Negative correlation was found in levels of IL-10 (P=0,041), IL-12 (P=0,038) and IL-1B (P=0,026). When cytokine levels of plasma samples with detectable and undetectable FVIII inhibitor titers were compared, significant higher plasma levels of TNFa (median: 11,56pg/ml, 8,11pg/ml; P=0,016) and lower levels of IL-12 (median: 4,29 pg/ml, 6,25 pg/ml; P=0,047) and IL-23 (median: 1016 pg/ml, 1353 pg/ml; P=0,049) were measured in samples with detectable FVIII inhibitor (BUpos). Furthermore, TNFa levels were higher in BUlow (median: 10,83 pg/ml; P=0,047) as well as in BUhigh samples (median: 11,75 pg/ml; P=0,019), compared to BUneg (median: 8,11 pg/ml). Cytokine concentrations of IL-1B (median: 2,64 pg/ml, 3,77 pg/ml; P=0,023), IL-2 (median: 2,44 pg/ml, 2,97pg/ml; P=0,043) and IL-17 (median: 15,79 pg/ml, 19,42 pg/ml; P=0,036) were significantly lower in BUhigh plasma samples compared to BUneg. Additionally, plasma level of IL-10 correlated negatively with levels of anti-FVIII IgG (P=0.045). Conclusion This is the first study of cytokine measurement in a longitudinal setting as well as during ITI in patients with hemophilia. FVIII inhibitors and anti-FVIII IgG antibodies were correlated to IL-10 and TNFa levels - of note, polymorphisms in the genes of these cytokines are a known risk factor for inhibitor development. Furthermore, IL-12, IL-17 and IL-23 levels were higher in samples with loss of FVIII Inhibitors. In addition to prediction of inhibitor development, cytokine profiles might serve as prognostic factors for ITI success and considering the emerging evidence of the IL-17-IL-23 immune axis in autoimmunity might also be promising therapeutic approaches for higher ITI success rates. Disclosures Ewing: Genentech: Honoraria; Shire: Honoraria; Bayer: Honoraria; Grifols: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Hema Biologics: Honoraria; Biogen: Research Funding. Koenigs:Jansen: Research Funding; Gilead: Research Funding; Biotest: Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Intersero: Research Funding; CSL Behring: Consultancy, Research Funding; EU (IMI, FP7): Research Funding; Sobi: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding; Novo Nordisk: Consultancy, Speakers Bureau; Bioverativ: Consultancy; Roche/Chugai: Consultancy.
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- 2018
17. Prospective Evaluation of the Thrombotic Risk in Children With Acute Lymphoblastic Leukemia Carrying the MTHFR TT 677 Genotype, the Prothrombin G20210A Variant, and Further Prothrombotic Risk Factors
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Nowak-Göttl, Ulrike, Wermes, Cornelia, Junker, Ralf, Koch, Hans-Georg, Schobess, Rosmarie, Fleischhack, Gudrun, Schwabe, Dirk, and Ehrenforth, Silke
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- 1999
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18. Mutational Landscape, Clonal Evolution Patterns and Role of RAS Mutations in Relapsed Acute Lymphoblastic Leukemia
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Oshima, Koichi, primary, Khiabanian, Hossein, additional, da Silva Almeida, Ana Carolina, additional, Tzoneva, Gannie, additional, Abate, Francesco, additional, Ambesi-Impiombato, Alberto, additional, Sanchez-Martin, Marta, additional, Carpenter, Zachary, additional, Penson, Alexander, additional, Perez-Garcia, Arianne, additional, Eckert, Cornelia, additional, Nicolás, Concepción, additional, Balbin, Milagros, additional, Sulis, Maria Luisa, additional, Kato, Motohiro, additional, Koh, Katsuyoshi, additional, Paganin, Maddalena, additional, Basso, Giuseppe, additional, Gastier-Foster, Julie M., additional, Devidas, Meenakshi, additional, Loh, Mignon L., additional, Kirschner-Schwabe, Renate, additional, Palomero, Teresa, additional, Rabadan, Raul, additional, and Ferrando, Adolfo A., additional
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- 2016
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19. Identification of an Ultra High-Risk and Targetable Molecular Signature in Relapsed Pediatric T-ALL
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Richter-Pechanska, Paulina, primary, Kunz, Joachim B., additional, Hof, Jana, additional, Zimmermann, Martin, additional, Rausch, Tobias, additional, Bandapalli, Obul Reddy, additional, Orlova, Elena, additional, Scapinello, Greta, additional, Sagi, Judit, additional, Stanulla, Martin, additional, Schrappe, Martin, additional, Kirschner-Schwabe, Renate, additional, Eckert, Cornelia, additional, Benes, Vladimir, additional, Korbel, Jan O., additional, Muckenthaler, Martina U., additional, and Kulozik, Andreas E, additional
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- 2016
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20. Multi-Genomics of Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia Reveals Three Distinct Genetic Clusters Characterized By Different Alterations
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Schroeder, Michael P, primary, Neumann, Martin, additional, Eckert, Cornelia, additional, Bastian, Lorenz, additional, James, Alva Rani, additional, Gökbuget, Nicola, additional, Schlee, Cornelia, additional, Ortiz Tanchez, Jutta, additional, Isaakidis, Konstandina, additional, Schwartz, Stefan, additional, Burmeister, Thomas, additional, Hoelzer, Dieter, additional, Pfeiffer, Heike, additional, Rieger, Michael A, additional, Göllner, Stefanie, additional, Oellerich, Thomas, additional, Yepes, Diego, additional, Kirschner-Schwabe, Renate, additional, Brüggemann, Monika, additional, Müller-Tidow, Carsten, additional, Serve, Hubert, additional, and Baldus, Claudia D., additional
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- 2016
- Full Text
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21. Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a
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Brettschneider, Kerstin, primary, Schmidt, Anja, additional, Kahle, Joerg, additional, Orlowski, Aleksander, additional, Stichel, Diana, additional, Becker-Peters, Karin, additional, Neimanis, Sonja, additional, Heller, Christine, additional, Klingebiel, Thomas, additional, Schwabe, Dirk, additional, and Koenigs, Christoph, additional
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- 2016
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22. Subclonal NT5C2mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia
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Barz, Malwine J., Hof, Jana, Groeneveld-Krentz, Stefanie, Loh, Jui Wan, Szymansky, Annabell, Astrahantseff, Kathy, vonStackelberg, Arend, Khiabanian, Hossein, Ferrando, Adolfo A., Eckert, Cornelia, and Kirschner-Schwabe, Renate
- Abstract
Activating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2mutations in relapsed ALL, we analyzed NT5C2in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2mutations compared with those without (19% and 25% vs 53%, P< .001). However, subclonal, but not clonal, NT5C2mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P= .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P< .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.
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- 2020
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23. Thromboembolic events in children with acute lymphoblastic leukemia (BFM protocols): prednisone versus dexamethasone administration
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Elvira Ahlke, Rosemarie Schobess, Dirk Schwabe, Christiane Schumann, Gudrun Fleischhack, Ulrike Nowak-Göttl, and Ralf Junker
- Subjects
Male ,medicine.medical_specialty ,Vincristine ,Adolescent ,medicine.drug_class ,Immunology ,Thrombophilia ,Biochemistry ,Gastroenterology ,Dexamethasone ,Risk Factors ,Prednisone ,Thromboembolism ,Acute lymphocytic leukemia ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,Coagulopathy ,Asparaginase ,Humans ,Medicine ,Risk factor ,Child ,business.industry ,Daunorubicin ,Infant ,Cell Biology ,Hematology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,medicine.disease ,Surgery ,Child, Preschool ,Corticosteroid ,business ,medicine.drug - Abstract
Alterations in hemostasis leading to symptomatic thromboembolism have been observed in patients with acute lymphoblastic leukemia (ALL) receiving Escherichia coli asparaginase (CASP) combined with steroids. Moreover, hereditary prothrombotic risk factors are associated with an increased risk for venous thromboembolism in pediatric ALL patients treated according to the BFM 90/95 protocols (including CASP combined with prednisone during induction therapy). To assess whether the thromboembolic risk associated with established prothrombotic risk factors is modified by treatment modalities (prednisone or dexamethasone), the present analysis was performed. Three hundred thirty-six consecutively recruited leukemic children treated according to different BFM protocols (PRED group, n = 280, 60 mg/m2 prednisone; DEXA group, n = 56, 10 mg/m2 dexamethasone during induction therapy) were studied. Study end point was the onset of symptomatic vascular accidents during induction therapy. Cumulative thromboembolism-free survival was significantly reduced in children in the PRED group (thrombosis frequency, 10.4%) compared with children in the DEXA group (thrombosis frequency, 1.8%; P = .028). Although no significant difference was found in the overall prevalence of prothrombotic risk factors, 46.5% of patients in the PRED group who experienced thromboembolic events were carriers of a prothrombotic risk factor, whereas no carrier in the DEXA group had a thromboembolism. At the time of maximum CASP activity, fibrinogen and activities of antithrombin, plasminogen, and protein S were significantly reduced in the PRED group. No significant correlation could be found between CASP activity and levels of coagulation factors. In conclusion, the use of dexamethasone instead of prednisone, administered with CASP, significantly reduced the onset of venous thromboembolism.
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- 2003
24. Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition
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Christina Halsey, Arend von Stackelberg, Stefanie Groeneveld-Krentz, Jana Hof, Lynne Minto, Isabella Swidenbank, Helen J. Blair, Elizabeth Matheson, Christine J. Harrison, Julie Irving, Marian Case, James M. Allan, Frida Ponthan, Josef Vormoor, Cornelia Eckert, and Renate Kirschner-Schwabe
- Subjects
Neuroblastoma RAS viral oncogene homolog ,Immunology ,Mice, Transgenic ,Mice, SCID ,medicine.disease_cause ,Biochemistry ,Mice ,Gene Frequency ,Mice, Inbred NOD ,Recurrence ,Acute lymphocytic leukemia ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Child ,Protein Kinase Inhibitors ,Mutation ,Clinical Trials as Topic ,Lymphoid Neoplasia ,business.industry ,Wild type ,Cell Biology ,Hematology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,medicine.disease ,MAP Kinase Kinase Kinases ,Xenograft Model Antitumor Assays ,PTPN11 ,Genes, ras ,Ras Signaling Pathway ,Drug Resistance, Neoplasm ,Cancer research ,Selumetinib ,Benzimidazoles ,KRAS ,business ,Signal Transduction - Abstract
For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.
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- 2014
25. Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a
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Christine Heller, Diana Stichel, Dirk Schwabe, Sonja Neimanis, Joerg Kahle, Thomas Klingebiel, Anja Schmidt, Karin Becker-Peters, Aleksander Orlowski, Kerstin Brettschneider, and Christoph Koenigs
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congenital, hereditary, and neonatal diseases and abnormalities ,education.field_of_study ,biology ,business.industry ,animal diseases ,ELISPOT ,Immunology ,Population ,Cell Biology ,Hematology ,Biochemistry ,medicine.anatomical_structure ,Antigen ,Immunotoxin ,hemic and lymphatic diseases ,Monoclonal ,medicine ,biology.protein ,Pseudomonas exotoxin ,Antibody ,education ,business ,B cell - Abstract
The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice. As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein. To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response. In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
26. Identification of an Ultra High-Risk and Targetable Molecular Signature in Relapsed Pediatric T-ALL
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Paulina Richter-Pechanska, Martin Zimmermann, Judit C. Sági, Greta Scapinello, Jana Hof, Martin Schrappe, Cornelia Eckert, Martina U. Muckenthaler, Obul Reddy Bandapalli, Martin Stanulla, Renate Kirschner-Schwabe, Joachim B. Kunz, Andreas E. Kulozik, Vladimir Benes, Jan O. Korbel, Tobias Rausch, and Elena Orlova
- Subjects
Oncology ,Mutation rate ,medicine.medical_specialty ,dbSNP ,business.industry ,Immunology ,Copy number analysis ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Chromosome 17 (human) ,Leukemia ,Internal medicine ,medicine ,Missense mutation ,Multiplex ligation-dependent probe amplification ,business - Abstract
Relapsed T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology as it is often resistant to treatment and fatal. In the search for genes that define critical steps of relapse and can serve as prognostic markers we compared 67 relapses (REL) and 147 samples collected at initial diagnosis (INI) by targeted sequencing of 313 leukemia-related genes. In addition to the analysis of single nucleotide variants (SNV) and small insertions and deletions (InDels), we made use of the available coverage data for profiling of copy number alterations (CNA). Of the 147 INI patients, 31 were treated according to the ALL-BFM 2000 and 116 according to the AIEOP-BFM ALL 2009 protocol. All REL patients were recruited from the ALL-REZ BFM 2002 trial. We analyzed bone marrow DNA by targeted capture of 313 genes (5964 exons) using the Haloplex Target Enrichment Kit (Agilent). We used Varscan to detect both SNV and InDels. In the absence of available remission samples, we subtracted known SNPs (dbSNP, 1000 gp) and variants present in at least one of 20 non-leukemic samples that we sequenced in parallel to the patients' samples. Only mutations with an AF >10% were considered. Copy number analysis based on read-depth data was validated by MLPA analysis of 14 genes showing a sensitivity of 99% and by low coverage WGS. In total, we have SNV data on all 147 INI and 67 REL patients and CNA data on 144 and 58 patients, respectively. Altogether, we identified relapse specific genetic events in 32 of 67 RELs. Our results confirm that NT5C2 mutations are highly enriched in relapse (REL: 17/67 vs. INI: 1/147; p=0.0001). Although activation of NT5C2 was associated with the occurrence of early first relapse (p=0.02), it did not correlate with induction failure and therefore has no prognostic impact. Similarly, amplifications of chromosome 17 q11.2-24.3, a region that contains genes of the STAT and ABCA families were significantly more frequent in REL than in INI (REL 7/58 vs. INI 3/144, p=0.0068), but also had no prognostic implications. By contrast, TP53 mutations were highly predictive of a second event: all 8 patients who carried a total of 9 TP53 mutations and deletions died within 9 months after first relapse (p=0.002), whereas 17 of the other 58 (29%) survived. Inactivation of TP53 was significantly correlated with higher mutation rates in other genes compared to those with wild-type TP53 (ttest= p In conclusion, targeted sequencing of relapsed T-ALL identified a molecular signature predicting an exquisitely poor outcome in 50% of relapses, who failed salvage treatment. This group of patients does not benefit from current treatment strategies thus identifying a subgroup with a dire clinical need for experimental therapy. Notably, approx. 25% REL patients who failed induction carried RAS and IL7R mutations. These patients may be considered for personalized treatment with either MEK- or JAK/STAT-inhibitors. Another 4 patients carry TP53 missense mutations and may benefit from treatment with p53 refolding compounds. Disclosures No relevant conflicts of interest to declare.
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- 2016
27. Multi-Genomics of Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia Reveals Three Distinct Genetic Clusters Characterized By Different Alterations
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Claudia D. Baldus, Diego Yepes, Cornelia Eckert, Hubert Serve, Renate Kirschner-Schwabe, Thomas Burmeister, Michael P Schroeder, Cornelia Schlee, Carsten Müller-Tidow, Stefan Schwartz, Lorenz Bastian, Alva Rani James, Dieter Hoelzer, Monika Brüggemann, Nicola Gökbuget, Stefanie Göllner, Michael A. Rieger, Heike Pfeiffer, Jutta Ortiz Tanchez, Martin Neumann, Thomas Oellerich, and Konstandina Isaakidis
- Subjects
0301 basic medicine ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Minimal residual disease ,Loss of heterozygosity ,03 medical and health sciences ,ETV6 ,030104 developmental biology ,0302 clinical medicine ,Differentially methylated regions ,CDKN2A ,030220 oncology & carcinogenesis ,Chromosome instability ,Acute lymphocytic leukemia ,CDKN2B ,medicine - Abstract
Introduction: Despite the recent identification of the Ph-like subgroup of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), a large number of BCP-ALL patients lack cytogenetic and molecular defined lesions. To get a higher resolution and a broader molecular view of relapsed BCP-ALL, we designed a multi-omics study to reveal age-overriding relapse-driving alterations that may unravel novel molecular targets. Methods: We studied 150 paired samples (initial diagnosis: ID; relapse: REL; complete remission: CR) from 50 patients without known translocations. The cohort consisted of 24 adult and 26 pediatric patients with minimal residual disease < 0.05 % at CR. All patients were treated in population based German study trials (GMALL, BFM). We examined the mutational and copy number status via exome sequencing, obtained expression profiles and fusion-genes via RNA-sequencing and the methylation status via Illumina Methylation Array. Results: With a lenient approach detecting drivers and passengers, we identified significantly more mutations in REL compared to ID samples (adult median: 52 vs 38; pediatric median: 39 vs 27). In addition, we detected 4 hypermutators (more than 100 mutations per sample), 2 were pediatric and 2 were adult samples, 3 of which were REL samples. The most recurrently mutated genes were KRAS (n=15), NRAS (n=15), TP53 (n=13), CDC27 (n=13), KMT2D (n=11), IKZF1 (n=11), CREBBP (n=10) and FLT3 (n=6; Figure 1), with mutations present in both age cohorts. NT5C2, SYK and CHD1 were exclusively mutated in the pediatric cohort with at least 3 mutations. NT5C2 was also specific for early REL. Of all REL mutations, 225 mutations (14%, mean: 4 mutations/patient) were sub-clonal (under < 5% mutation frequency) at ID. Copy number alterations (CNA) varied greatly among pediatric and adult samples: 6% of pediatric and 18% of adult samples had aneuploidies and or copy neutral loss of heterozygosity of whole chromosomes. Chromosomal aberrations at ID persisted at relapse (100 %). Particular targets of CNA affected well-described genes like CDKN2A, CDKN2B, PAX5 on chr9p. Genes preferentially subjected to homozygous deletions were VPREB1 (n=6), SH2B3 (n=4), and ETV6 (n=2). All SH3B2 deletions were found in pediatric samples. On the epi-genomic level, the principal component analysis of the most variable CG-sites revealed a stable methylation profile during the course of the disease. However, we found a clear separation into a smaller pediatric-dominated cluster (n=24; 20 pediatric, 4 adult) and a larger mixed-age cluster (n=76; Fig. 1, Cluster A). Differentially methylated regions, affecting a total of 269 genes, characterized the separation of the smaller cluster, henceforth called Methylation Deregulated (MDR) cluster. The samples of the MDR cluster showed also a distinct gene expression profile by RNA-seq supporting a tight connection between the methylation status and its transcriptional program. A subset of 97 genes was differentially expressed including MAPK and PDGFR genes as most prominently deregulated. Additionally we defined a MDR expression classifier comprising 30 genes (Fig. 1). On the mutational level, the MDR samples had 20 % fewer mutations (mean: 25.3) compared to the remaining samples (mean: 31.3) and fewer CNVs for the most frequently affected genes. Characterising the non-MDR samples, a third of those were categorized as Ph-like ALL using the 15 gene classifier in an unsupervised clustering; this signature also coincided with the presence of well-known fusion-genes (Fig. 1, Cluster B). The remaining samples were defined by chromosomal instability (CI; Fig. 1, Cluster C). In the CI cluster, mutations in epigenetic regulators were twice as frequent when compared to the remaining samples. Conclusions: We describe three distinct clusters in relapsed BCP-ALL, which are characterized by a different genetic alterations: a novel MDR cluster by distinct methylation changes, the Ph-like cluster by gene fusions and the CI cluster by chromosomal instability. The cluster assignment was stable over the course of the disease. All clusters occurred in pediatric and adult patients, with the methylation-driven cluster predominantly in pediatrics. The MDR cluster showed significantly fewer mutations and CNVs compared to the other two clusters. The MDR samples showed activation of the MAPK signaling pathway pointing to actionable therapeutic targets. Figure 1 Figure 1. Disclosures Gökbuget: Pfizer: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.
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- 2016
28. Mutational Landscape, Clonal Evolution Patterns and Role of RAS Mutations in Relapsed Acute Lymphoblastic Leukemia
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Mignon L. Loh, Giuseppe Basso, Adolfo A. Ferrando, Katsuyoshi Koh, Renate Kirschner-Schwabe, Motohiro Kato, Francesco Abate, Teresa Palomero, Arianne Perez-Garcia, Concepcion Nicolas, Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Gannie Tzoneva, Koichi Oshima, Meenakshi Devidas, Julie M. Gastier-Foster, Maddalena Paganin, Zachary Carpenter, Milagros Balbín, Raul Rabadan, Hossein Khiabanian, Maria Luisa Sulis, Cornelia Eckert, Alex Penson, and Ana C. da Silva-Almeida
- Subjects
0301 basic medicine ,Neuroblastoma RAS viral oncogene homolog ,Vincristine ,Immunology ,Biology ,medicine.disease_cause ,Biochemistry ,Somatic evolution in cancer ,Clonal Evolution ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Acute lymphocytic leukemia ,medicine ,Humans ,Childhood Acute Lymphoblastic Leukemia ,Multidisciplinary ,Base Sequence ,Wild type ,Combination chemotherapy ,Cell Biology ,Hematology ,Biological Sciences ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,medicine.disease ,Virology ,PTPN11 ,Leukemia ,Genes, ras ,Methotrexate ,030104 developmental biology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Mutation ,Cancer research ,KRAS ,medicine.drug - Abstract
Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children. Altogether 90% of pediatric ALL patients achieve a complete hematologic remission with current high dose combination chemotherapy and 80% of them remain leukemia free. However, the outcome for patients showing refractory disease or those whose leukemia relapses after an initial transient response remains disappointingly poor with cure rates of less than 40%. To investigate genetic drivers of relapse and resistance and explore the specific roles of clonal evolution in disease progression and relapse here we performed whole-exome sequence analysis of matched diagnosis, germline (remission) and relapse DNA samples in a panel of 55 pediatric ALL patients including 33 T-cell ALLs and 22 B-cell precursor ALLs. These analyses identified an average of 9 mutations present in diagnostic samples and 17 mutations in relapsed leukemia DNAs. Phylogenetic tree analysis for each of the 48 cases with optimal variant call parameters analyzing their clonal evolution dynamics during disease progression, combined with whole genome sequencing of targeted samples with low exonic mutation input, showed that branched evolution in which relapse clones contain some, but not all genetic lesions present in the major clone at diagnosis as the primary mechanism driving tumor progression and relapse present in 45/48 (94%) cases. In addition, and consistent with previous reports we identified the presence of chemotherapy associated mutations in NT5C2 (10/55), TP53 (3/55), CREBBP (4/55) and the NR3C1 glucocorticoid receptor gene (2/55). However, and most strikingly, 23/27 (85%) recurrently mutated genes in this series with mutations preferentially selected or retained at the time of relapse (mutation never lost in the relapse clone) were not implicated in relapse ALL before (HTR3A, MED12, USP9X, CACNA1H, ODZ3, AACS, SAMD4A, ANO5, PAPPA, NAALADL2, HIST3H2A, FZD7, TBX15, NEB, GREB1L, PLXNA4, SGK223, TSC1, PTPRG, FGF10, SYCP2, TRPM3 and EYS). A branched pattern of genetic evolution and the presence of recurrent mutations selected at relapse support that chemotherapy imposes a strong Darwinian genetic selection in leukemic cell populations. In this context it is worth noting that RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, while in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Most notably, and in agreement with this hypothesis, inducible expression of mutant KRAS in human ALL lines demonstrate that oncogenic KRAS G12D induces methotrexate resistance, but also improves leukemia response to vincristine; a phenotype perfectly recapitulated in a isogenic ALL leukemia model generated from a conditional inducible Kras G12D knockin mice. Mechanistically, KRAS G12 expression induces MAPK dependent abrogation of methotrexate induced apoptosis. Moreover, Kras mutant tumors show enhanced G2/M cell cycle arrest and apoptosis upon spindle poisoning with vincristine, a phenotype linked with increased PLK phosphorylation and transcriptional down-regulation of mitotic genes. Finally clonal competition assays demonstrate that the differential response to methotrexate and vincristine in isogenic Kras wild type and Kras mutant ALL cells results in clonal dominance of Kras G12D populations in cultures treated with methotrexate, while Kras wild type cells are selected the context of vincristine treatment. In all these results show novel insight on the genetics and mechanisms of clonal selection, disease progression and relapse in ALL and demonstrate a previously unrecognized dual role of RAS mutations in chemotherapy response. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.
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- 2016
29. Prospective Evaluation of the Thrombotic Risk in Children With Acute Lymphoblastic Leukemia Carrying the MTHFR TT 677 Genotype, the Prothrombin G20210A Variant, and Further Prothrombotic Risk Factors
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C. Wermes, Ralf Junker, Dirk Schwabe, Gudrun Fleischhack, Ulrike Nowak-Göttl, Rosmarie Schobess, Hans Georg Koch, and Silke Ehrenforth
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medicine.medical_specialty ,education.field_of_study ,biology ,business.industry ,Antithrombin ,Population ,Immunology ,Factor V ,Cell Biology ,Hematology ,medicine.disease ,Gastroenterology ,Biochemistry ,Protein C deficiency ,Internal medicine ,Methylenetetrahydrofolate reductase ,biology.protein ,medicine ,Prothrombin G20210A ,Risk factor ,education ,business ,Protein C ,medicine.drug - Abstract
The reported incidence of thromboembolism in children with acute lymphoblastic leukemia (ALL) treated with L-asparaginase, vincristine, and prednisone varies from 2.4% to 11.5%. The present study was designed to prospectively evaluate the role of the TT677 methylenetetrahydrofolate reductase (MTHFR) genotype, the prothrombin G20210A mutation, the factor V G1691A mutation, deficiencies of protein C, protein S, antithrombin, and increased lipoprotein (a) concentrations in leukemic children treated according to the ALL-Berlin-Frankfurt-Muenster (BFM) 90/95 study protocols with respect to the onset of vascular events. Three hundred and one consecutive leukemic children were enrolled in this study. Fifty-five of these 301 subjects investigated had one established single prothrombotic risk factor: 20 children showed the TT677 MTHFR genotype; 5 showed the heterozygous prothrombin G20210A variant; 11 were carriers of the factor V G1691A mutation (heterozygous, n = 10; homozygous, n = 1); 4 showed familial protein C, 4 protein S, and 2 antithrombin type I deficiency; 9 patients were suffering from familially increased lipoprotein (a) [Lp(a)] concentrations (>30 mg/dL). In addition, combined prothrombotic defects were found in a further 10 patients: the FV mutation was combined with the prothrombin G20210A variant (n = 1), increased Lp(a) (n = 3), protein C deficiency (n = 1), and homozygosity for the C677T MTHFR gene mutation (n = 1). Lp(a) was combined with protein C deficiency (n = 2) and the MTHFR TT 677 genotype (n = 2). Two hundred eighty-nine of the 301 patients were available for thrombosis-free survival analysis. In 32 (11%) of these 289 patients venous thromboembolism occurred. The overall thrombosis-free survival in patients with at least one prothrombotic defect was significantly reduced compared with patients without a prothrombotic defect within the hemostatic system (P < .0001). In addition, a clear-cut positive correlation (P < .0001) was found between thrombosis and the use of central lines. However, because the prothrombotic defects diagnosed in the total childhood population studied were all found within the prevalences reported for healthy Caucasian individuals, the interaction between prothrombotic risk factors, ALL treatment, and further environmental factors is likely to cause thrombotic manifestations.
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- 1999
30. De Novo Purine Biosynthesis in Drug Resistance and Tumor Relapse of Childhood ALL
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Li, Hui, primary, Li, Benshang, additional, Yang, Fan, additional, Duan, Caiwen, additional, Bai, Yun, additional, Yang, Jun J, additional, Chen, Jing, additional, von Stackelberg, Arend, additional, Chen, Hongzhuan, additional, Tang, JingYan, additional, Ferrando, Adolfo A., additional, Zhang, Jinghui, additional, Wang, Shengyue, additional, Kirschner-Schwabe, Renate, additional, and Zhou, Bin-Bing S., additional
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- 2015
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31. Gene Panel Sequencing of Primary and Relapsed Pediatric T-ALL Shows That Relapse-Specific Mutations Are Diverse and Mostly Non-Recurrent
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Pechanska, Paulina, primary, Kunz, Joachim, additional, Rausch, Tobias, additional, Bandapalli, Obul Reddy, additional, Orlova, Elena, additional, Sagi, Judit, additional, Stanulla, Martin, additional, Schrappe, Martin, additional, Kirschner-Schwabe, Renate, additional, Hof, Jana, additional, Eckert, Cornelia, additional, Köhler, Rolf, additional, Muckenthaler, Martina U., additional, Korbel, Jan o., additional, and Kulozik, Andreas E, additional
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- 2015
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32. Frequency and Epitope Specificity of Anti-Factor VIII C1 Domain Antibodies in Acquired and Hereditary Hemophilia Inhibitor Patient Plasma
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Kahle, Joerg, primary, Orlowski, Aleksander, additional, Schmidt, Anja, additional, Brettschneider, Kerstin, additional, Klingebiel, Thomas, additional, Healey, John F, additional, Parker, Ernest T, additional, Lollar, John (Pete) S, additional, Jacquemin, Marc, additional, Krause, Manuela, additional, Tiede, Andreas, additional, Schwabe, Dirk, additional, and Königs, Christoph, additional
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- 2015
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33. TEL-AML1 Fusion Transcript in Relapsed Childhood Acute Lymphoblastic Leukemia
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Dirk Buchwald, Birgit Beyermann, Karlheinz Seeger, Günter Henze, Bernhard Kremens, Hans-Peter Adams, Jörg Ritter, Dörte Harms, Dirk Schwabe, Charlotte M. Niemeyer, and Martin Schrappe
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Oncology ,medicine.medical_specialty ,business.industry ,Incidence (epidemiology) ,Immunology ,Chromosomal translocation ,Cell Biology ,Hematology ,Philadelphia chromosome ,medicine.disease ,Biochemistry ,Surgery ,Leukemia ,medicine.anatomical_structure ,Fusion transcript ,Acute lymphocytic leukemia ,Internal medicine ,hemic and lymphatic diseases ,medicine ,Bone marrow ,business ,Childhood Acute Lymphoblastic Leukemia - Abstract
The cryptic translocation t(12;21)(p13;q22) has been recently recognized as the most common genetic rearrangement in B-lineage childhood acute lymphoblastic leukemia (ALL). The resulting fusion transcript, termed TEL-AML1, has been associated with an excellent prognosis at initial ALL diagnosis. Hence, we postulated that the incidence of TEL-AML1 fusion should be lower in patients with ALL relapse. To address this assumption and to investigate the prognostic significance of TEL-AML1 expression in relapsed childhood ALL, bone marrow samples of 146 children were analyzed by reverse-transcriptase (RT)-polymerase chain reaction (PCR). All children were treated according to Berlin-Frankfurt-Münster (BFM) ALL relapse trial protocols (ALL-REZ BFM 90-96). Their clinical features and outcome were compared with those of 262 patients who could not be tested due to lack of bone marrow samples. Thirty-two of 146 children with relapsed ALL were TEL-AML1–positive. Four of the negative patients had T-lineage and nine Philadelphia chromosome (Ph1)-positive leukemia. Thus, the incidence ofTEL-AML1 in relapsed Ph1-negative, B-cell precursor ALL is 32 of 133 (24%). The 32 TEL-AML1–positive and 101 negative patients differed significantly with respect to duration of last remission (42.5 v 27 months; P = .0001) and age at initial diagnosis (53.5 v 74 months;P = .0269). At a median follow-up time of 21.5 months, children positive for TEL-AML1 had a significantly (P = .0011) higher probability of event-free survival (EFS; 0.79 v 0.33). The predominant majority of patients had been treated for initial ALL according to German multicenter BFM (108 of 133) or Cooperative ALL study group (CoALL) (19 of 133) frontline protocols. The comparison of tested and not-tested (N = 262) patients showed no significant difference.TEL-AML1 positivity predicted a favorable short-term outcome; long-term results are unknown. Screening for TEL-AML1 should become routine at relapse diagnosis and might be used for therapy stratification in future trials.
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- 1998
34. De Novo Purine Biosynthesis in Drug Resistance and Tumor Relapse of Childhood ALL
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Jingyan Tang, Yun Bai, Fan Yang, Renate Kirschner-Schwabe, Jinghui Zhang, Cai-Wen Duan, Arend von Stackelberg, Bin-Bing S. Zhou, Benshang Li, Hui Li, Jun J. Yang, Adolfo A. Ferrando, Jing Chen, Hong-Zhuan Chen, and Shengyue Wang
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Inosine monophosphate ,chemistry.chemical_classification ,Mutation ,Immunology ,Combination chemotherapy ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,Biochemistry ,Molecular biology ,De novo synthesis ,chemistry.chemical_compound ,chemistry ,Lometrexol ,medicine ,Nucleotide ,Purine metabolism ,Hypoxanthine - Abstract
Background: Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Studies have shown that most ALL cases are polyclonal at diagnosis and that genetic changes in individual subclones influence sensitity to therapy and subsequent clonal evolution during therapy; but the molecular details remain to be worked out. Among different pathways enriched for mutations at relapse, purine metabolism is particularly interesting for two reasons: first, thiopurines are widely used in the ALL combination chemotherapy regimens, and are prodrugs that are converted by the purine salvage pathway to cytotoxic metabolites. Second, de novo nucleotide biosynthesis is often upregulated in cancer cells, and it is believed that sufficient nucleotide pools are required to maintain genomic stability, could bypass oncogene-induced senescence and promote tumor progression1. Therefore, we focus our current study on de novo purine biosynthesis in drug resistance and tumor relapse of childhood ALL. Methods and Results: Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. Targeted sequencing identified mutations in additional genes in de novo purine biosynthesis pathway, providing further genetic evidence for its importance in relapsed ALL. All individuals with PRPS1 mutation relapsed early on-treatment (P Using various functional assays, we demonstrated that rather than causing a simple gain-of-function effect, the mutations in PRPS1 resulted in the disruption of the normal feedback inhibition of purine synthesis, in which the enzyme remained active despite an increased concentration of nucleoside analogs. PRPS1 mutants increased synthesis of the nucleoside inosine monophosphate, its metabolite hypoxanthine (HX) and de novo purine biosynthesis intermediates (e.g. AICAR, SAICAR) in Reh cells. Increased intracellular HX can competively inhibit the conversion of thiopurines into their active metabolites. Furthermore, inhibition of de novo purine biosynthesis in vitro, either by CRISPR-Cas9 genome editing of de novo purine synthesis pathway genes (GART, ATIC etc.) or treatment with a pathway inhibitor lometrexol (GART inhibitor) alleviated the metabolic disturbance and drug resistance induced by PRPS1 mutations. Using ultra-deep sequencing of unique serial remission samples before clinical relapse, we noticed that the PRPS1 mutant allele fraction increased drastically before clinical relapse, suggesting rapid clonal expansion occurs after the acquisition of a PRPS1 mutation. Interestingly, we also noticed that PPRS1 mutation coexist with RAS mutation in many relapse cases and at single cell resolution. Functional analysis revealed that tumor cells which harbored RAS and PRPS1 double mutations are more drug resistant than those with RAS or PRPS1 mutation alone. Previous studies have shown that oncogenic RAS mutation can also induce various stress responses including oncogene-induced senensence and DNA damage response (DDR), which all could impede tumor cell proliferation during relapse. In vitro, we found PRPS1 mutation can release the replication and metabolic stress caused by RAS mutation, in addition to their role in thiopurine resistance. The PRPS1 mutants not only increase the nucleotide pools but also elevate purine biosynthesis intermediate AICAR, which can activate AMPK and reduce the RAS mutant-induced DDR. We are currently working on in vitro and in vivo models (including patient derived xenograft models) to further test the double mutant's effects on tumor-reinitiation and clonal evolution during ALL relapse. Conclusions: We demonstrated that negative feedback-defective PRPS1 mutants can drive de novo purine biosynthesis, which can exert drug resistance and reduce genomic instability during tumor relapse. Our study highlights the importance of de novo purine biosynthesis in the pathogenesis of relapse, and suggests a diagnostic approach to predicting early relapse and a therapeutic strategy to circumventing resistance in ALL. 1 Li et al. Negative feedback-defective PRPS1 mutants drivee thiopurine resistance in relapsed childhood ALL. Nature Medicine,21(6): 563-571 (2015) Disclosures No relevant conflicts of interest to declare.
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- 2015
35. Gene Panel Sequencing of Primary and Relapsed Pediatric T-ALL Shows That Relapse-Specific Mutations Are Diverse and Mostly Non-Recurrent
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Jana Hof, Jan O. Korbel, Cornelia Eckert, Martina U. Muckenthaler, Judit C. Sági, Rolf Köhler, Tobias Rausch, Renate Kirschner-Schwabe, Obul Reddy Bandapalli, Elena Orlova, Joachim B. Kunz, Andreas E. Kulozik, Martin Schrappe, Paulina Pechanska, and Martin Stanulla
- Subjects
Point mutation ,Immunology ,Cell Biology ,Hematology ,Biology ,MAP3K7 ,Biochemistry ,Molecular biology ,Exon ,CDKN2A ,CDKN2B ,Multiplex ligation-dependent probe amplification ,Allele frequency ,Exome sequencing - Abstract
Precursor T-cell acute lymphoblastic leukemia (T-ALL) remains one of the major challenges of pediatric oncology, because relapses are frequently refractory to treatment and fatal. We aimed at identifying relapse specific genetic alterations by analyzing a cohort of 147 primary T-ALL patients and of 70 relapsed T-ALL patients with targeted gene panel sequencing. In addition to the analysis of single nucleotide variants (SNVs) and small insertions and deletions (InDels), we made use of the available coverage data to characterize aberrant copy number alterations (CNA). DNA from bone marrow of these 217 pediatric T-ALL patients was analyzed by gene panel sequencing after target capture with Agilent HaloPlex. In the target capture design, exons of 324 genes were included that had been found before by whole exome sequencing to carry somatic mutations in a pilot set of relapsed T-ALL or that have been reported to be mutated in T-ALL in the literature. We did not analyze corresponding remission samples and did not discriminate between germline and somatic alterations. Only mutations with an allele frequency (AF) > 10% were considered and absence of the mutation in the 1000 Genomes variant catalogue was required. Copy number analysis based on read-depth data identified deletions (DEL) and amplifications (AMP). Direct comparison of CNAs by multiplex ligation-dependent probe amplification (MPLA) and gene panel sequencing was possible for 13 overlapping regions covering 14 genes in 185 samples. Recognition rate by coverage analysis was 98% (256/260) for biallelic alterations and 81% (92/114) for monoallelic alterations found by MLPA. On average, gene panel sequencing identified 6.7 mutations in initial diagnosis samples (SNVs: 5.2; InDels: 1.5) and 7.9 mutations in relapse samples (SNVs: 5.9; InDels: 2). In the group of primary leukemia and relapse samples, the average AMP/DEL per patient was 8.2 (AMP: 3.2, DEL: 5.0) and 8.8 (AMP: 4.2, DEL: 4.6), respectively. 31 genes were found to be mutated and 46 deleted/amplified in 10 or more patients (see Table 1 and 2). | Gene | Total # of mutations | # pts with mutation in primary T-ALL (n=147) | # pts with mutation in relapsed T-ALL (n=70) | | ------ | -------------------- | --------------------------------------------------- | -------------------------------------------- | | NOTCH1 | 178 | 88 (60%) | 38 (54%) | | PHF6 | 47 | 24 (16%) | 16 (23%) | | FBXW7 | 42 | 20 (14%) | 17 (24%) | | OBSCN | 35 | 20 (14%) | 10 (14%) | | DNM2 | 28 | 17 (12%) | 10 (14%) | | PTEN | 34 | 20 (14%) | 4 (6%) | | XIRP2 | 24 | 19 (13%) | 5 (7%) | | CDH23 | 23 | 11 (7%) | 11 (16%) | | WT1 | 36 | 11 (7%) | 8 (11%) | | NT5C2 | 22 | 1 (1%) | 17 (24%) | Table 1. Most commonly mutated genes (SNVs and InDels) | Amplifications | Deletions | | -------------- | --------------------- | --------------------- | | Gene | primary T-ALL (n=147) | relapsed T-ALL (n=64) | Gene | primary T-ALL (n=147) | relapsed T-ALL (n=64) | | MYB | 9 (6%) | 9 (15%) | CDKN2A | 102 (70%) | 36 (59%) | | MYC | 11 (8%) | 6 (10%) | CDKN2B | 83 (57%) | 31 (51%) | | NRG1 | 11 (8%) | 3 (5%) | MLLT3 | 28 (19%) | 5 (8%) | | UNC5D | 11 (8%) | 3 (5%) | PHIP | 18 (12%) | 6 (10%) | | NCOA2 | 11 (8%) | 3 (5%) | ELOVL4 | 18 (12%) | 5 (8%) | | PTK2B | 11 (8%) | 3 (5%) | MAP3K7 | 17 (12%) | 5 (8%) | | FDFT1 | 11 (8%) | 3 (5%) | CASP8AP2 | 17 (12%) | 5 (8%) | | ABL1 | 8 (5%) | 3 (5%) | APC | 19 (13%) | 3 (5%) | | CNOT3 | 8 (5%) | 3 (5%) | LEF1 | 16 (11%) | 5 (8%) | | SMG8 | 3 (2%) | 7 (10%) | PAX5 | 15 (10%) | 5 (8%) | Table 2. Most common copy number alterations Potential novel mechanisms of oncogene activation are amplifications of PTK2B, a gene that has been found to be deregulated by fusion in Philadelphia-like BCP-ALL and that is potentially targetable by tyrosine kinase inhibitors, and of MYC, which has long been known to be a key player in T-ALL leukemogenesis and that is amplified in neuroblastoma and medulloblastoma. Enriched in relapse, we identified mutations in NT5C2 (p=1.4E-08), TP53 (p=0.0006) and CCDC88A (p=0.01), and amplifications of a region on chr 17q represented by the genes CLTC, ABCA5, C17orf80 and SRSF2. MLLT3 deletions were enriched in primary samples (p=0.04), consistent with the observation that MLLT3 deletions confer a lower risk of relapse in patients treated on BFM protocols. Conclusion Gene panel sequencing emerges as a suitable tool for a comprehensive genetic characterization of pediatric T-ALL. Within the group of selected genes contained in the panel, CNA were as frequent as point mutations. Only few genes were found to be specifically altered in relapse, indicating that progression to relapse may involve diverse, non-recurrent genetic alterations. Disclosures No relevant conflicts of interest to declare.
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- 2015
36. Frequency and Epitope Specificity of Anti-Factor VIII C1 Domain Antibodies in Acquired and Hereditary Hemophilia Inhibitor Patient Plasma
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Andreas Tiede, Anja Schmidt, Marc Jacquemin, John S. Lollar, Joerg Kahle, Manuela Krause, John F. Healey, Thomas Klingebiel, Kerstin Brettschneider, Ernest T. Parker, Dirk Schwabe, Aleksander Orlowski, and Christoph Königs
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education.field_of_study ,business.operation ,biology ,business.industry ,medicine.drug_class ,Immunology ,Population ,Cell Biology ,Hematology ,Octapharma ,Monoclonal antibody ,Biochemistry ,Virology ,Epitope ,Epitope mapping ,Monoclonal ,Mutation testing ,biology.protein ,Medicine ,Antibody ,business ,education - Abstract
Acquired hemophilia A (AHA) is a rare autoimmune disease caused by development of inhibitory anti-factor VIII (fVIII) antibodies (also called inhibitors) resulting in severe hemorrhages. In addition, inhibitor development is the most serious complication of today's replacement therapy in patients with hereditary X-linked hemophilia A (HA) disorder. Earlier studies showed that antibodies in AHA and HA inhibitor plasmas are both primarily directed to the A2 and C2 domains suggesting that these two domains are the predominant immunogenic fVIII regions (Fulcher et al, 1987; Prescott et al, 1997; Lollar, 2004). However, the C1 domain also makes a major contribution to the humoral anti-fVIII immune response in hemophilic mice (Healey et al, 2007), which motivated us to analyze the frequency and epitope specificity of anti-C1 antibodies in AHA and HA inhibitor patient plasma. The frequency of domain-specific antibodies were studied by antibody binding to human A2, C1 and C2 domains presented as (i) single human domain (SHD) human/porcine hybrid fVIII and (ii) HSA-fusion proteins. While similar frequencies of A2- and C2-specific antibodies were observed for both applied mapping strategies the use of isolated C1 domain resulted in much higher detection level of anti-C1 antibodies compared to the use of the human C1 domain human/porcine hybrid fVIII protein. As homologue-scanning mutagenesis relies on differences among human and porcine sequences these results suggest the presence of a large number of cross-reactive anti-C1 antibodies binding to species-conserved epitopes. Overall, anti-C1 antibodies were detected in 90 of 115 (78%) AHA and 36 of 63 (57%) HA inhibitor patients. Two well-characterized monoclonal C1 inhibitors, human LE2E9 (Jacquemin et al, 2000) and murine MAb 2A9 (ASH 2014 poster, Batsuli et al) were used for indirect epitope mapping of anti-C1 antibodies in AHA patients by competition binding studies. Our results for AHA patients with non-crossreactive anti-C1 antibodies only (n=11) show that antibody binding to human C1 domain human/porcine hybrid fVIII (HP53) protein was completely blocked in the presence of MAb 2A9. In contrast, antibody binding to the isolated C1 domain was only partially reduced in the presence of MAb 2A9 for a selected number of (high responding) AHA patients (n=10) suggesting the presence of a second population of crossreactive anti-C1 antibodies that exclusively bind to conserved amino acid residues. Competition binding to native and denatured fVIII and HP53 proteins revealed that MAb 2A9 and LE2E9 bind mutually exclusive to a conformational C1 epitope involving amino acid residues that are not conserved between humans and pigs. Consequently, essential binding residues were identified for both C1 inhibitors via the use of HP53 variants, in which surface exposed non-conserved amino acid residues on the human C1 domain were substituted for porcine residues. The results of this mutational analysis showed that despite their competitive binding different amino acid residues are essential for binding of MAb 2A9 and LE2E9. These findings are in agreement with the different specific inhibitory activities of the two C1 inhibitors (97 BU/mg vs 10000 BU/mg). Finally, HSA-C1 point mutants were used to directly map essential epitope residues of anti-C1 antibodies in AHA and HA inhibitor patient plasma. Our study demonstrates that a large number of AHA and HA inhibitor patients (126 of 178; 71%) have anti-C1 antibodies that comprise at least two different populations, crossreactive and non-crossreactive to porcine fVIII. Therefore, in addition to the A2 and C2 domains, the C1 domain seems to significantly contribute to the immune response to fVIII in these patients. As recent data point toward a functional role of the fVIII C1 domain for membrane-, fX-, and von Willebrand factor-binding (Lü et al, 2011) the clinical relevance of anti-C1 antibodies should be analyzed in further studies. Disclosures Tiede: Leo Pharma: Consultancy, Honoraria; Novo Nordisk: Consultancy, Honoraria, Research Funding; Coachrom: Research Funding; SOBI: Consultancy, Honoraria; Biogen Idec: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Octapharma: Other: Investigator, Speakers Bureau; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Investigator, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Königs:Bayer: Research Funding, Speakers Bureau; Biotest: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Sobi: Consultancy; CSL Behring: Research Funding, Speakers Bureau; Intersero: Research Funding; NovoNordisk: Speakers Bureau.
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- 2015
37. The gamma subunit of the interleukin-2 receptor is expressed in human monocytes and modulated by interleukin-2, interferon gamma, and transforming growth factor beta 1
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MC Bosco, I Espinoza-Delgado, M Schwabe, SM Russell, WJ Leonard, DL Longo, and L Varesio
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
The interleukin-2 receptor gamma (IL-2R gamma) chain is a newly recognized component of the IL-2R of lymphoid cells that is required for their response to IL-2. We investigated the expression of IL-2R gamma protein in human monocytes by Western blot analysis using an antiserum specific for IL-2R gamma. We found that IL-2R gamma subunit is constitutively expressed in human monocytes and upregulated by the monocyte-activating factors IL-2 and interferon gamma (IFN gamma). Furthermore, we show that transforming growth factor beta 1 (TGF beta 1) downmodulates, in a dose-dependent manner, basal and IL-2-induced, but not IFN gamma-induced, IL-2R gamma chain expression, and this effect may be responsible for TGF beta 1 suppressive activity on IL-2- activated monocytes. Overall, these results show that the expression of the IL-2R gamma subunit in human monocytes is tightly regulated by the cytokine network, suggesting a critical role played by this protein on monocyte activation.
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- 1994
38. The gamma subunit of the interleukin-2 receptor is expressed in human monocytes and modulated by interleukin-2, interferon gamma, and transforming growth factor beta 1
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Luigi Varesio, Michael Schwabe, Maria Carla Bosco, Warren J. Leonard, Dan L. Longo, Igor Espinoza-Delgado, and Sarah M. Russell
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Interleukin 2 ,medicine.medical_specialty ,Monocyte ,Immunology ,Cell Biology ,Hematology ,Transforming growth factor beta ,Biology ,Biochemistry ,Molecular biology ,Endocrinology ,medicine.anatomical_structure ,Cell surface receptor ,Internal medicine ,medicine ,biology.protein ,Interferon gamma ,TGF beta 1 ,medicine.drug ,Gamma subunit ,Common gamma chain - Abstract
The interleukin-2 receptor gamma (IL-2R gamma) chain is a newly recognized component of the IL-2R of lymphoid cells that is required for their response to IL-2. We investigated the expression of IL-2R gamma protein in human monocytes by Western blot analysis using an antiserum specific for IL-2R gamma. We found that IL-2R gamma subunit is constitutively expressed in human monocytes and upregulated by the monocyte-activating factors IL-2 and interferon gamma (IFN gamma). Furthermore, we show that transforming growth factor beta 1 (TGF beta 1) downmodulates, in a dose-dependent manner, basal and IL-2-induced, but not IFN gamma-induced, IL-2R gamma chain expression, and this effect may be responsible for TGF beta 1 suppressive activity on IL-2- activated monocytes. Overall, these results show that the expression of the IL-2R gamma subunit in human monocytes is tightly regulated by the cytokine network, suggesting a critical role played by this protein on monocyte activation.
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- 1994
39. Regulation by interleukin-2 (IL-2) and interferon gamma of IL-2 receptor gamma chain gene expression in human monocytes
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Maria Carla Bosco, Igor Espinoza-Delgado, Dan L. Longo, Michael Schwabe, K Sugamura, Luigi Varesio, and G L Gusella
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Interleukin 2 ,Messenger RNA ,Monocyte ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,medicine.anatomical_structure ,Cytokine ,Gene expression ,medicine ,Interferon gamma ,IL-2 receptor ,medicine.drug ,Common gamma chain - Abstract
The interleukin-2 receptor gamma chain (IL-2R gamma) gene codes for a subunit of the IL-2R and is expressed in human lymphoid cells. The present study was undertaken to determine whether human monocytes expressed the IL-2R gamma gene constitutively or after activation by IL- 2 or interferon gamma (IFN gamma). Fresh human monocytes constitutively expressed low but significant levels of IL-2R gamma mRNA, and nuclear run-on experiments showed that IL-2R gamma gene was transcriptionally active. Stimulation with IL-2 or IFN gamma induced a major increase of IL-2R gamma mRNA in a time- and a dose-dependent manner. However, neither cytokine increased the transcriptional activity of the gene. The enhancement of IL-2R gamma mRNA expression by either IL-2 or IFN gamma was concomitant with the stabilization of the mRNA, suggesting a postranscriptional level of control. Finally, the augmented expression of IL-2R gamma in IL-2- and IFN gamma-treated monocytes was associated with an increased IL-2-binding activity, compared with that of unstimulated cells. These results provide the first evidence of the expression of the IL-2R gamma gene in nonlymphoid cells and of its modulation by IL-2 and IFN gamma through posttranscriptional mechanisms.
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- 1994
40. Elevated lipoprotein(a) concentration is an independent risk factor of venous thromboembolism
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Ulrike Nowak-Göttl, Ralf Junker, Dirk Schwabe, Karin Kurnik, Rosemarie Schobess, and Gudrun Fleischhack
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medicine.medical_specialty ,Asparaginase ,biology ,Vascular disease ,business.industry ,Deep vein ,Immunology ,Cell Biology ,Hematology ,Lipoprotein(a) ,medicine.disease ,Biochemistry ,Thrombosis ,chemistry.chemical_compound ,medicine.anatomical_structure ,Endocrinology ,Embolism ,chemistry ,Internal medicine ,medicine ,biology.protein ,Cardiology ,Risk factor ,business ,Venous thromboembolism - Abstract
We have read with great interest the letter “Increased lipoprotein(a) levels are not a steady prothrombotic defect” by Korte et al, recently published in Blood .[1][1] The authors report on a small case series of 7 pediatric patients treated with asparaginase either because of acute
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- 2002
41. Relapsing Pediatric T-Cell Acute Lymphoblastic Leukemia Downregulates T-Cell Properties and Upregulates Cell Adhesion
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Richter-Pechanska, Paulina, Kunz, Joachim B., Rausch, Tobias, Von Knebel Doeberitz, Caroline, Frismantas, Viktoras, Dobay, Maria Pamela, Bornhauser, Beat, Zimmermann, Martin, Bandapalli, Obul Reddy, Fuhrmann, Stephan, Orlova, Elena, Uhrig, Sebastian, Balasubramanian, Gnana Prakash, Stanulla, Martin, Schrappe, Martin, Cario, Gunnar, Escherich, Gabriele, Bakharevich, Kseniya, Kirschner-Schwabe, Renate, Eckert, Cornelia, Pfister, Stefan, Korbel, Jan O., Muckenthaler, Martina, Bourquin, Jean-Pierre, and Kulozik, Andreas E.
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- 2017
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42. PDX Models Recapitulate Genetics and Epigenetics of Pediatric T-Cell Leukemia
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Von Knebel Doeberitz, Caroline, Richter-Pechanska, Paulina, Frismantas, Viktoras, Kunz, Joachim B., Rausch, Tobias, Bornhauser, Beat, Zimmermann, Martin, Bandapalli, Obul Reddy, Orlova, Elena, Seeman, Julia, Erarslan, Busra, Assenov, Yassen, Stanulla, Martin, Schrappe, Martin, Cario, Gunnar, Escherich, Gabriele, Bakharevich, Kseniya, Kirschner-Schwabe, Renate, Eckert, Cornelia, Korbel, Jan O., Muckenthaler, Martina, Bourquin, Jean-Pierre, and Kulozik, Andreas E.
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- 2017
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43. Clonal Evolution Mechanisms and Collateral Sensitivity to Impdh Inhibitor Therapy in Relapsed Lymphoblastic Leukemia
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Dieck, Chelsea, Tzoneva, Gannie, Oshima, Koichi, Ambesi-Impiombato, Alberto, Sanchez-Martin, Marta, Sulis, Maria Luisa, Kato, Motohiro, Koh, Katsuyoshi, Paganin, Maddalena, Basso, Giuseppe, Gastier-Foster, Julie M., Loh, Mignon L., Kirschner-Schwabe, Renate, Madubata, Chioma, Rabadan, Raul, and Ferrando, Adolfo A.
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- 2017
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44. Constitutional SAMD9L Mutations Cause Familial Myelodisplastic Syndrome with Monosomy 7 and Stable Revertant Mosaicism
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Pastor Loyola, Victor, Sahoo, Sushree Sangita, Boklan, Jessica, Schwabe, Georg, Strahm, Brigitte, Lebrecht, Dirk, Voss, Matthias, Bryceson, Yenan T, Erlacher, Miriam, Ehninger, Gerhard, Niewisch, Marena, Schlegelberger, Brigitte, Baumann, Irith, Achermann, John, Shimamura, Akiko, Hochrein, Jochen, Tedgaard, Ulf, Nilsson, Lars, Hasle, Henrik, Börries, Melanie, Busch, Hauke, Niemeyer, Charlotte M., and Wlodarski, Marcin W.
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- 2017
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45. CD11b is a therapy resistance- and minimal residual disease-specific marker in precursor B-cell acute lymphoblastic leukemia
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Rita Mitlohner, Leonid Karawajew, Giuseppe Basso, Renate Kirschner-Schwabe, Martin Schrappe, Christian Hagemeier, Martin Stanulla, Giuseppe Gaipa, Michael Dworzak, Richard Ratei, Peter Rhein, and Wolf-Dieter Ludwig
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Oncology ,Male ,medicine.medical_specialty ,Neoplasm, Residual ,Adolescent ,Immunology ,Antineoplastic Agents ,Biochemistry ,Bone Marrow ,Internal medicine ,Precursor cell ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,medicine ,Biomarkers, Tumor ,Neoplasm ,Humans ,Clinical significance ,Prospective Studies ,RNA, Messenger ,Child ,Survival rate ,Oligonucleotide Array Sequence Analysis ,B-Lymphocytes ,CD11b Antigen ,business.industry ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Remission Induction ,Infant ,Cell Biology ,Hematology ,medicine.disease ,Minimal residual disease ,Gene expression profiling ,Survival Rate ,medicine.anatomical_structure ,Treatment Outcome ,Drug Resistance, Neoplasm ,Child, Preschool ,Female ,Bone marrow ,business ,Burkitt's lymphoma - Abstract
A consistently increased mRNA expression of the adhesion receptor CD11b is a hallmark of the reported genomewide gene expression changes in precursor B-cell acute lymphoblastic leukemia (PBC-ALL) after 1 week of induction therapy. To investigate its clinical relevance, CD11b protein expression in leukemic blasts has been prospectively measured at diagnosis (159 patients) and during therapy (53 patients). The initially heterogeneous expression of CD11b inversely correlated with cytoreduction rates measured at clinically significant time points of induction therapy in the ALL–Berlin-Frankfurt-Münster 2000 protocol. CD11b positivity conferred a 5-fold increased risk of minimal residual disease (MRD) after induction therapy (day 33) and of high-risk group assignment after consolidation therapy (day 78). In the multivariate analysis CD11b expression was an independent prognostic factor compared with other clinically relevant parameters at diagnosis. During therapy, CD11b expression increased early in most ALL cases and remained consistently increased during induction/consolidation therapy. In more than 30% of MRD-positive cases, the CD11b expression on blast cells exceeded that of mature memory B cells and improved the discrimination of residual leukemic cells from regenerating bone marrow. Taken together, CD11b expression has considerable implications for prognosis, treatment response monitoring, and MRD detection in childhood PBC-ALL.
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- 2010
46. The AF4.MLL fusion protein is capable of inducing ALL in mice without requirement of MLL.AF4
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Theo Dingermann, Karen Schwabe, Brigitte Rüster, Rolf Marschalek, Reinhard Henschler, Martin Ruthardt, and A Bursen
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Male ,Oncogene Proteins ,Oncogene Proteins, Fusion ,Immunology ,Chromosomal translocation ,Biology ,Biochemistry ,Leukemogenic ,Translocation, Genetic ,Fusion gene ,Mice ,Transduction, Genetic ,hemic and lymphatic diseases ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,medicine ,Animals ,Humans ,neoplasms ,Regulation of gene expression ,Gene Expression Regulation, Leukemic ,Nuclear Proteins ,Cell Biology ,Hematology ,Histone-Lysine N-Methyltransferase ,medicine.disease ,Fusion protein ,Chromosomes, Mammalian ,Transplantation ,DNA-Binding Proteins ,Leukemia ,Cell Transformation, Neoplastic ,Cancer research ,Myeloid-Lymphoid Leukemia Protein - Abstract
The chromosomal translocation t(4;11)(q21;q23) is the most frequent genetic aberration of the human MLL gene, resulting in high-risk acute lymphoblastic leukemia (ALL). To elucidate the leukemogenic potential of the fusion proteins MLL·AF4 and AF4·MLL, Lin−/Sca1+ purified cells (LSPCs) were retrovirally transduced with either both fusion genes or with MLL·AF4 or AF4·MLL alone. Recipients of AF4·MLL- or double-transduced LSPCs developed pro-B ALL, B/T biphenotypic acute leukemia, or mixed lineage leukemia. Transplantation of MLL·AF4- or mock-transduced LSPCs did not result in disease development during an observation period of 13 months. These findings indicate that the expression of the AF4·MLL fusion protein is capable of inducing acute lymphoblastic leukemia even in the absence of the MLL·AF4 fusion protein. In view of recent findings, these results may imply that t(4;11) leukemia is based on 2 oncoproteins, providing an explanation for the very early onset of disease in humans.
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- 2010
47. Clinical Significance of NT5C2 Mutations in Children with First Relapse of B-Cell Precursor Acute Lymphoblastic Leukemia
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Hof, Jana, primary, Szymansky, Annabell, additional, von Stackelberg, Arend, additional, Eckert, Cornelia, additional, and Kirschner-Schwabe, Renate, additional
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- 2014
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48. Targeted Deep Sequencing of Genetic Alterations Identified By Whole Exome Sequencing Reveals Clonal Evolution in Pediatric T-Lymphoblastic Leukemia
- Author
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Kunz, Joachim, primary, Bandapalli, Obul R, additional, Rausch, Tobias, additional, Stuetz, Adrian, additional, Pechanska, Paulina, additional, Assenov, Yassen, additional, Eilers, Juliane, additional, Orlova, Elena, additional, Sagi, Judit, additional, Stanulla, Martin, additional, Schrappe, Martin, additional, Handgretinger, Rupert, additional, Kirschner-Schwabe, Renate, additional, Hof, Jana, additional, Eckert, Cornelia, additional, Avigad, Smadar, additional, Koehler, Rolf, additional, Muckenthaler, Martina U., additional, Korbel, Jan, additional, and Kulozik, Andreas E, additional
- Published
- 2014
- Full Text
- View/download PDF
49. Functional Analysis of Phage Display Selected Single-Chain Variable Antibody Fragments (scFvs) Specific for Anti-FVIII Antibodies
- Author
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Brettschneider, Kerstin, primary, Naumann, Anja, additional, Neimanis, Sonja, additional, Kahle, Joerg, additional, Heller, Christine, additional, Klingebiel, Thomas, additional, Schwabe, Dirk, additional, and Koenigs, Christoph, additional
- Published
- 2014
- Full Text
- View/download PDF
50. The Prognostic Impact of the Mutational Profile in Patients with Myelofibrosis in the Era of the JAK1/JAK2-Inhibitor Ruxolitinib
- Author
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Al-Ali, Haifa Kathrin, primary, Wang, Song-Yau, additional, Jaekel, Nadja, additional, Koehler, Anne, additional, Krahl, Rainer, additional, Hubert, Karolin, additional, Lange, Thoralf, additional, Roskos, Martin, additional, Stengel, Rayak, additional, Kovacs, Ines, additional, Scarlett, Schwabe, additional, Schubert, Karoline, additional, Wildenberger, Kathrin, additional, Schneider, Melanie, additional, Koehler, Elisabeth, additional, and Niederwieser, Dietger, additional
- Published
- 2014
- Full Text
- View/download PDF
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