48 results on '"Evelyn, Zellmeier"'
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2. Supplementary Figures from Evolution of Cytogenetically Normal Acute Myeloid Leukemia During Therapy and Relapse: An Exome Sequencing Study of 50 Patients
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Karsten Spiekermann, Wolfgang Hiddemann, Bernhard J. Wörmann, Wolfgang E. Berdel, Dennis Görlich, Stephan Wolf, Stefan K. Bohlander, Claudia D. Baldus, Martin Neumann, Helmut Blum, Stefan Krebs, Alexander Graf, Stephanie Schneider, Nikola P. Konstandin, Bianka Ksienzyk, Evelyn Zellmeier, Kathrin Bräundl, Friederike Pastore, Daniela Schumacher, Vindi Jurinovic, Paul Kerbs, Stefanos A. Bamopoulos, Tobias Herold, Klaus H. Metzeler, Ines Hellmann, Raphael Mattes, Sophie M. Stief, Sebastian Vosberg, Luise Hartmann, and Philipp A. Greif
- Abstract
Figure S1 Recurrently mutated genes and comparison to TCGA cohort. Figure S2 Stabililty of recurrently mutated genes over disease course. Figure S3 Variant allele frequency plots from diagnosis to relapse for each individual patient. Figure S4 Mutation patterns of individual genes. Lines represent mutations in individual patients. Figure S5 Detection of the pre-existence of gained variants at initial diagnosis. Figure S6 Mutations in AML-associated functional pathways. Figure S7 Deletion of exons 3-10 of the KDM6A gene in the MM-6 cell line. The sister cell line MM-1 is not affected. Deletions were detected by quantitative MLPA analysis and the peak ratio for each KDM6A exon specific probe is shown. Graph represents the results from two independent experiments. Figure S8 H3K27 methylation in the AML cell lines MM-1 and MM-6. The global mono-, di- and tri-methylation of H3K27 in MM-1 and MM-6 was analyzed by Western blot and normalized to H3. The median of three independent experiments is shown. P-Values were calculated using a two-tailed, unpaired Student's t-test. Figure S9 The MM-6 cell line is resistant to cytarabine (Ara-C) but not to Daunorubicin or AC220. Error bars indicate mean {plus minus} s.d. of at least three independent experiments. **P75th percentile) with clinical outcome according to gender in the AMLCG-99 trial (NCT00266136). Figure S11 Proportion of transversions from diagnosis to relapse. Transversion frequencies are shown for lost (blue), stable (orange) and gained (red) mutations. Dots represent individual patients. Figure S12 Median coverage in targeted sequencing of persistent and non-persistent DNMT3A variant positions at complete remission (CR). Figure S13 Clinical outcome according to mutation persistence at remission.
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- 2023
3. Data from Evolution of Cytogenetically Normal Acute Myeloid Leukemia During Therapy and Relapse: An Exome Sequencing Study of 50 Patients
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Karsten Spiekermann, Wolfgang Hiddemann, Bernhard J. Wörmann, Wolfgang E. Berdel, Dennis Görlich, Stephan Wolf, Stefan K. Bohlander, Claudia D. Baldus, Martin Neumann, Helmut Blum, Stefan Krebs, Alexander Graf, Stephanie Schneider, Nikola P. Konstandin, Bianka Ksienzyk, Evelyn Zellmeier, Kathrin Bräundl, Friederike Pastore, Daniela Schumacher, Vindi Jurinovic, Paul Kerbs, Stefanos A. Bamopoulos, Tobias Herold, Klaus H. Metzeler, Ines Hellmann, Raphael Mattes, Sophie M. Stief, Sebastian Vosberg, Luise Hartmann, and Philipp A. Greif
- Abstract
Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations.Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters.Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse.Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.
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- 2023
4. ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation
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Luise Hartmann, Sayantanee Dutta, Sabrina Opatz, Sebastian Vosberg, Katrin Reiter, Georg Leubolt, Klaus H. Metzeler, Tobias Herold, Stefanos A. Bamopoulos, Kathrin Bräundl, Evelyn Zellmeier, Bianka Ksienzyk, Nikola P. Konstandin, Stephanie Schneider, Karl-Peter Hopfner, Alexander Graf, Stefan Krebs, Helmut Blum, Jan Moritz Middeke, Friedrich Stölzel, Christian Thiede, Stephan Wolf, Stefan K. Bohlander, Caroline Preiss, Linping Chen-Wichmann, Christian Wichmann, Maria Cristina Sauerland, Thomas Büchner, Wolfgang E. Berdel, Bernhard J. Wörmann, Jan Braess, Wolfgang Hiddemann, Karsten Spiekermann, and Philipp A. Greif
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Science - Abstract
The t(8;21) translocation is often found in acute myeloid leukaemia but is not sufficient for development of the disease. In this study, the authors identify frequent mutations in the transcriptional repressor, ZBTB7A, in these patients and show that the mutations reduce DNA binding activity.
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- 2016
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5. Adults with Philadelphia chromosome–like acute lymphoblastic leukemia frequently have IGH-CRLF2 and JAK2 mutations, persistence of minimal residual disease and poor prognosis
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Tobias Herold, Stephanie Schneider, Klaus H. Metzeler, Martin Neumann, Luise Hartmann, Kathryn G. Roberts, Nikola P. Konstandin, Philipp A. Greif, Kathrin Bräundl, Bianka Ksienzyk, Natalia Huk, Irene Schneider, Evelyn Zellmeier, Vindi Jurinovic, Ulrich Mansmann, Wolfgang Hiddemann, Charles G. Mullighan, Stefan K. Bohlander, Karsten Spiekermann, Dieter Hoelzer, Monika Brüggemann, Claudia D. Baldus, Martin Dreyling, and Nicola Gökbuget
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Philadelphia-like B-cell precursor acute lymphoblastic leukemia (Ph-like ALL) is characterized by distinct genetic alterations and inferior prognosis in children and younger adults. The purpose of this study was a genetic and clinical characterization of Ph-like ALL in adults. Twenty-six (13%) of 207 adult patients (median age: 42 years) with B-cell precursor ALL (BCP-ALL) were classified as having Ph-like ALL using gene expression profiling. The frequency of Ph-like ALL was 27% among 95 BCP-ALL patients negative for BCR-ABL1 and KMT2A-rearrangements. IGH-CRLF2 rearrangements (6/16; P=0.002) and mutations in JAK2 (7/16; P
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- 2017
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6. Molecular response assessment by quantitative real-time polymerase chain reaction after induction therapy in NPM1-mutated patients identifies those at high risk of relapse
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Max Hubmann, Thomas Köhnke, Eva Hoster, Stephanie Schneider, Annika Dufour, Evelyn Zellmeier, Michael Fiegl, Jan Braess, Stefan K. Bohlander, Marion Subklewe, Maria-Cristina Sauerland, Wolfgang E. Berdel, Thomas Büchner, Bernhard Wörmann, Wolfgang Hiddemann, and Karsten Spiekermann
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Monitoring minimal residual disease is an important way to identify patients with acute myeloid leukemia at high risk of relapse. In this study we investigated the prognostic potential of minimal residual disease monitoring by quantitative real-time polymerase chain reaction analysis of NPM1 mutations in patients treated in the AMLCG 1999, 2004 and 2008 trials. Minimal residual disease was monitored - in aplasia, after induction therapy, after consolidation therapy, and during follow-up - in 588 samples from 158 patients positive for NPM1 mutations A, B and D (with a sensitivity of 10−6). One hundred and twenty-seven patients (80.4%) achieved complete remission after induction therapy and, of these, 56 patients (44.1%) relapsed. At each checkpoint, minimal residual disease cut-offs were calculated. After induction therapy a cut-off NPM1 mutation ratio of 0.01 was associated with a high hazard ratio of 4.26 and the highest sensitivity of 76% for the prediction of relapse. This was reflected in a cumulative incidence of relapse after 2 years of 77.8% for patients with ratios above the cut-off versus 26.4% for those with ratios below the cut-off. In the favorable subgroup according to European LeukemiaNet, the cut-off after induction therapy also separated the cohort into two prognostic groups with a cumulative incidence of relapse of 76% versus 6% after 2 years. Our data demonstrate that in addition to pre-therapeutic factors, the course of minimal residual disease in an individual is an important prognostic factor and could be included in clinical trials for the guidance of post-remission therapy. The trials from which data were obtained were registered at www.clinicaltrials.gov (#NCT01382147, #NCT00266136) and at the European Leukemia Trial Registry (#LN_AMLINT2004_230).
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- 2014
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7. The NPM1 mutation type has no impact on survival in cytogenetically normal AML.
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Friederike Pastore, Philipp A Greif, Stephanie Schneider, Bianka Ksienzyk, Gudrun Mellert, Evelyn Zellmeier, Jan Braess, Cristina M Sauerland, Achim Heinecke, Utz Krug, Wolfgang E Berdel, Thomas Buechner, Bernhard Woermann, Wolfgang Hiddemann, and Karsten Spiekermann
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Medicine ,Science - Abstract
NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML.
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- 2014
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8. Evolution of Cytogenetically Normal Acute Myeloid Leukemia During Therapy and Relapse: An Exome Sequencing Study of 50 Patients
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Tobias Herold, Luise Hartmann, Wolfgang Hiddemann, Philipp A. Greif, Daniela Schumacher, Bianka Ksienzyk, Claudia D. Baldus, Bernhard Wörmann, Evelyn Zellmeier, Stephan Wolf, Sebastian Vosberg, Friederike Pastore, Helmut Blum, Stephanie Schneider, Sophie M. Stief, Stefanos A. Bamopoulos, Wolfgang E. Berdel, Ines Hellmann, Stefan Krebs, Alexander Graf, Karsten Spiekermann, Klaus H. Metzeler, Stefan K. Bohlander, Vindi Jurinovic, Nikola P. Konstandin, Raphael Mattes, Dennis Görlich, Martin Neumann, Kathrin Bräundl, and Paul Kerbs
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Adult ,Male ,0301 basic medicine ,Oncology ,Cancer Research ,medicine.medical_specialty ,Myeloid ,Drug Resistance ,medicine.disease_cause ,Cell Line ,Epigenesis, Genetic ,Cytogenetics ,Young Adult ,03 medical and health sciences ,Recurrence ,Internal medicine ,Exome Sequencing ,Humans ,Medicine ,Exome ,DNA (Cytosine-5-)-Methyltransferases ,Epigenetics ,Young adult ,Exome sequencing ,Aged ,Aged, 80 and over ,Histone Demethylases ,Mutation ,business.industry ,Remission Induction ,Cytarabine ,Cancer ,Middle Aged ,medicine.disease ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,medicine.anatomical_structure ,Female ,business ,medicine.drug - Abstract
Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations. Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters. Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse. Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.
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- 2018
9. Acute myeloid leukemia with del(9q) is characterized by frequent mutations ofNPM1,DNMT3A, WT1and low expression ofTLE4
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Wolfgang Hiddemann, Stephanie Schneider, Alexander Graf, Sebastian Vosberg, Sabrina Opatz, Helmut Blum, Klaus H. Metzeler, Philipp A. Greif, Tobias Herold, Evelyn Zellmeier, Bernhard Wörmann, Karsten Spiekermann, Stefan Krebs, Nikola P. Konstandin, Luise Hartmann, Bianka Ksienzyk, Vindi Jurinovic, Maria Cristina Sauerland, Ulrich Mansmann, Wolfgang E. Berdel, Thomas Büchner, and Stefan K. Bohlander
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0301 basic medicine ,Genetics ,Cancer Research ,NPM1 ,Myeloid leukemia ,Karyotype ,Chromosome 9 ,Biology ,Genetic analysis ,Pathogenesis ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Cancer research ,Gene ,Exome sequencing - Abstract
Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P
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- 2016
10. Persistence of pre-leukemic clones during first remission and risk of relapse in acute myeloid leukemia
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Wolfgang Hiddemann, Maja Rothenberg-Thurley, Susanne Amler, Stefan K. Bohlander, Stephanie Schneider, Max Hubmann, Karsten Spiekermann, Bianka Ksienzyk, Maria Cristina Sauerland, Jan Braess, Klaus H. Metzeler, Andreas Faldum, Tobias Herold, Dennis Goerlich, Nikola P. Konstandin, Thomas Köhnke, Marion Subklewe, and Evelyn Zellmeier
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0301 basic medicine ,Oncology ,Adult ,Male ,Cancer Research ,medicine.medical_specialty ,Allogeneic transplantation ,Myeloid ,Neoplasm, Residual ,Risk Assessment ,Article ,DNA Methyltransferase 3A ,Clonal Evolution ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Bone Marrow ,Recurrence ,Internal medicine ,Biomarkers, Tumor ,Medicine ,Humans ,Cumulative incidence ,DNA (Cytosine-5-)-Methyltransferases ,Risk factor ,Aged ,Proportional Hazards Models ,Aged, 80 and over ,business.industry ,Hazard ratio ,Remission Induction ,Myeloid leukemia ,Hematology ,Middle Aged ,medicine.disease ,Combined Modality Therapy ,Transplantation ,Leukemia ,Leukemia, Myeloid, Acute ,030104 developmental biology ,medicine.anatomical_structure ,Treatment Outcome ,030220 oncology & carcinogenesis ,Mutation ,Female ,business ,Biomarkers - Abstract
Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ≥1 mutation during remission at a VAF of ≥2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (p
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- 2017
11. Adults with Philadelphia chromosome–like acute lymphoblastic leukemia frequently have IGH-CRLF2 and JAK2 mutations, persistence of minimal residual disease and poor prognosis
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Claudia D. Baldus, Ulrich Mansmann, Luise Hartmann, Tobias Herold, Kathryn G. Roberts, Irene Schneider, Dieter Hoelzer, Karsten Spiekermann, Charles G. Mullighan, Nicola Gökbuget, Evelyn Zellmeier, Natalia Huk, Wolfgang Hiddemann, Philipp A. Greif, Klaus H. Metzeler, Martin Dreyling, Bianka Ksienzyk, Martin Neumann, Kathrin Bräundl, Monika Brüggemann, Nikola P. Konstandin, Stephanie Schneider, Vindi Jurinovic, and Stefan K. Bohlander
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Adult ,Male ,medicine.medical_specialty ,Pediatrics ,Neoplasm, Residual ,Adolescent ,DNA Copy Number Variations ,Oncogene Proteins, Fusion ,Chromosomal translocation ,600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit ,Philadelphia chromosome ,Gastroenterology ,Translocation, Genetic ,Persistence (computer science) ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,Internal medicine ,medicine ,Cluster Analysis ,Humans ,Neoplasm ,ddc:610 ,Receptors, Cytokine ,Young adult ,Survival analysis ,Gene Rearrangement ,business.industry ,Gene Expression Profiling ,Articles ,Hematology ,Gene rearrangement ,Janus Kinase 2 ,Middle Aged ,Prognosis ,medicine.disease ,Survival Analysis ,Minimal residual disease ,Gene Expression Regulation, Neoplastic ,030220 oncology & carcinogenesis ,Mutation ,Female ,Immunoglobulin Heavy Chains ,business ,030215 immunology - Abstract
Philadelphia-like B-cell precursor acute lymphoblastic leukemia (Ph-like ALL) is characterized by distinct genetic alterations and inferior prognosis in children and younger adults. The purpose of this study was a genetic and clinical characterization of Ph-like ALL in adults. Twenty-six (13%) of 207 adult patients (median age: 42 years) with B-cell precursor ALL (BCP-ALL) were classified as having Ph-like ALL using gene expression profiling. The frequency of Ph-like ALL was 27% among 95 BCP-ALL patients negative for BCR- ABL1 and KMT2A-rearrangements. IGH-CRLF2 rearrangements (6/16; P=0.002) and mutations in JAK2 (7/16; P
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- 2017
12. Acute myeloid leukemia with del(9q) is characterized by frequent mutations of NPM1, DNMT3A, WT1 and low expression of TLE4
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Tobias, Herold, Klaus H, Metzeler, Sebastian, Vosberg, Luise, Hartmann, Vindi, Jurinovic, Sabrina, Opatz, Nikola P, Konstandin, Stephanie, Schneider, Evelyn, Zellmeier, Bianka, Ksienzyk, Alexander, Graf, Stefan, Krebs, Helmut, Blum, Maria, Cristina Sauerland, Thomas, Büchner, Wolfgang E, Berdel, Bernhard J, Wörmann, Ulrich, Mansmann, Wolfgang, Hiddemann, Stefan K, Bohlander, Karsten, Spiekermann, and Philipp A, Greif
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Adult ,Male ,Adolescent ,DNA Methyltransferase 3A ,Cohort Studies ,Young Adult ,Biomarkers, Tumor ,Humans ,Exome ,DNA (Cytosine-5-)-Methyltransferases ,WT1 Proteins ,Aged ,Neoplasm Staging ,Aged, 80 and over ,Chromosome Aberrations ,High-Throughput Nucleotide Sequencing ,Nuclear Proteins ,Middle Aged ,Prognosis ,Repressor Proteins ,Survival Rate ,Leukemia, Myeloid, Acute ,Mutation ,Female ,Chromosome Deletion ,Chromosomes, Human, Pair 9 ,Nucleophosmin ,Follow-Up Studies - Abstract
Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P 0.001, 8/14, 57% vs. 26/111, 23%; P = 0.02). Furthermore, we identified DNMT3B to be rarely but recurrently targeted by truncating mutations in AML. Gene expression analysis of 13 patients with del(9q) and 454 patients with normal karyotype or various cytogenetic aberrations showed significant down regulation of TLE4 in patients with del(9q) (P = 0.02). Interestingly, downregulation of TLE4 was not limited to AML with del(9q), potentially representing a common mechanism in AML pathogenesis. Our comprehensive genetic analysis of the del(9q) subgroup reveals a unique mutational profile with the frequency of DNMT3A mutations in the del(9q) only subset being the highest reported so far in AML, indicating oncogenic cooperativity. © 2016 Wiley Periodicals, Inc.
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- 2017
13. Inactivation of TP53 correlates with disease progression and low miR-34a expression in previously treated chronic lymphocytic leukemia patients
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Philippe Solal-Celigny, Martin Weisser, Ru-Fang Yeh, Tobias Benthaus, Gudrun Mellert, John Catalano, Wolfgang Hiddemann, Stefan K. Bohlander, Debraj GuhaThakurta, Guillemette Duchateau-Nguyen, Purvi M. Kakadia, Tadeusz Robak, Lin Wu, Evelyn Zellmeier, Marco Montillo, Sabine Lohmann, David Dornan, Nancy Patten, Christian H. Geisler, Sim Truong, Giuseppe Palermo, Stephanie Schneider, Jan Braess, Galina Salogub, Annika Dufour, Anna Dmoszynska, and Karsten Spiekermann
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Adult ,Male ,endocrine system diseases ,Chronic lymphocytic leukemia ,Immunology ,Down-Regulation ,Phases of clinical research ,Biochemistry ,Disease-Free Survival ,stomatognathic system ,Downregulation and upregulation ,Recurrence ,Biomarkers, Tumor ,Humans ,Medicine ,Gene Silencing ,Treatment Failure ,neoplasms ,Aged ,Regulation of gene expression ,medicine.diagnostic_test ,Gene Expression Regulation, Leukemic ,business.industry ,Point mutation ,Cell Biology ,Hematology ,Middle Aged ,Prognosis ,medicine.disease ,Leukemia, Lymphocytic, Chronic, B-Cell ,MicroRNAs ,Leukemia ,Disease Progression ,Cancer research ,Female ,Rituximab ,Tumor Suppressor Protein p53 ,business ,medicine.drug ,Fluorescence in situ hybridization - Abstract
In chronic lymphocytic leukemia (CLL) patients, disruptions of the TP53 tumor suppressor pathway by 17p13 deletion (del17p), somatic TP53 mutations, or downregulation of microRNA-34a have been associated with a poor prognosis. So far, the impact of the various TP53 defects has not been evaluated in a large cohort of previously treated and relapsed CLL patients. Here, we present the results of TP53 gene sequencing and fluorescence in situ hybridization for del17p in a phase 3 clinical trial (REACH [Rituximab in the Study of Relapsed Chronic Lymphocytic Leukemia]). Of the 457 patients, 52 had TP53 mutations and 37 had del17p. In 24 (46%) of the TP53 mutated patients, no del17p was found and in 9 of the del17p patients, no TP53 mutation was identified. Based on a predicted proportion of TP53 disruption, a complete disruption of TP53 function, either by a combination of point mutations and/or del17p, was associated with a high risk for disease progression. Progression-free survival of patients with a heterozygous TP53 mutation was not significantly different from patients with a completely intact TP53 locus. In addition, only a complete loss of TP53 function correlated with low microRNA-34a expression levels. This trial was registered at www.clinicaltrials.gov as #NCT00090051.
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- 2013
14. The new and recurrent FLT3 juxtamembrane deletion mutation shows a dominant negative effect on the wild-type FLT3 receptor
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Evelyn Zellmeier, Nikola P. Konstandin, Maja Rothenberg, Nadine Sandhöfer, Annika Dufour, Klaus H. Metzeler, Belay Tizazu, Karsten Spiekermann, Katrin Reiter, Wolfgang Hiddemann, Julia Bauer, Harald Polzer, and Philipp A. Greif
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Male ,0301 basic medicine ,Biology ,Tropomyosin receptor kinase C ,Article ,03 medical and health sciences ,fluids and secretions ,0302 clinical medicine ,Growth factor receptor ,Cell Line, Tumor ,hemic and lymphatic diseases ,Humans ,5-HT5A receptor ,Receptor ,Genes, Dominant ,Multidisciplinary ,hemic and immune systems ,Ligand (biochemistry) ,Molecular biology ,Leukemia, Myeloid, Acute ,030104 developmental biology ,fms-Like Tyrosine Kinase 3 ,030220 oncology & carcinogenesis ,Interleukin-21 receptor ,Mutation ,embryonic structures ,Fms-Like Tyrosine Kinase 3 ,ROR1 ,Female - Abstract
In acute myeloid leukemia (AML), the Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes. Recently, a new and recurrent juxtamembrane deletion mutation (p.Q569Vfs*2) resulting in a truncated receptor was identified. The mutated receptor is expressed on the cell surface and still binds its ligand but loses the ability to activate ERK signaling. FLT3 p.Q569fs-expressing Ba/F3 cells show no proliferation after ligand stimulation. Furthermore, coexpressed with the FLT3 wild-type (WT) receptor, the truncated receptor suppresses stimulation and activation of the WT receptor. Thus, FLT3 p.Q569Vfs*2, to our knowledge, is the first FLT3 mutation with a dominant negative effect on the WT receptor.
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- 2016
15. ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation
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Bianka Ksienzyk, Stephan Wolf, Karl-Peter Hopfner, Alexander Graf, Sabrina Opatz, Stefan Krebs, Helmut Blum, Maria Cristina Sauerland, Philipp A. Greif, Christian Wichmann, Jan Moritz Middeke, Evelyn Zellmeier, Bernhard Wörmann, Friedrich Stölzel, Wolfgang E. Berdel, Nikola P. Konstandin, Tobias Herold, Sebastian Vosberg, Stefanos A. Bamopoulos, Christian Thiede, Karsten Spiekermann, Linping Chen-Wichmann, Thomas Büchner, Georg Leubolt, Carolin Preiss, Klaus H. Metzeler, Katrin Reiter, Stephanie Schneider, Luise Hartmann, Jan Braess, Sayantanee Dutta, Wolfgang Hiddemann, Kathrin Bräundl, and Stefan K. Bohlander
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0301 basic medicine ,Myeloid ,Oncogene Proteins, Fusion ,Molecular biology ,Chromosomes, Human, Pair 21 ,General Physics and Astronomy ,Chromosomal translocation ,medicine.disease_cause ,600 Technik, Medizin, angewandte Wissenschaften::610 Medizin und Gesundheit ,Translocation, Genetic ,Fusion gene ,chemistry.chemical_compound ,RUNX1 Translocation Partner 1 Protein ,hemic and lymphatic diseases ,Cancer ,Mutation ,Multidisciplinary ,Gene Expression Regulation, Leukemic ,DNA-Binding Proteins ,Haematopoiesis ,Biological sciences ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,RUNX1 ,Core Binding Factor Alpha 2 Subunit ,Glycolysis ,Chromosomes, Human, Pair 8 ,Signal Transduction ,Lineage (genetic) ,Science ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Protein Domains ,Cell Line, Tumor ,medicine ,Genetics ,Humans ,Base Sequence ,Gene Expression Profiling ,RUNX1T1 ,General Chemistry ,Survival Analysis ,030104 developmental biology ,HEK293 Cells ,chemistry ,FOS: Biological sciences ,Transcription Factors - Abstract
The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion., The t(8;21) translocation is often found in acute myeloid leukaemia but is not sufficient for development of the disease. In this study, the authors identify frequent mutations in the transcriptional repressor, ZBTB7A, in these patients and show that the mutations reduce DNA binding activity.
- Published
- 2016
16. Acute Myeloid Leukemia With Biallelic CEBPA Gene Mutations and Normal Karyotype Represents a Distinct Genetic Entity Associated With a Favorable Clinical Outcome
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Wolfgang Hiddemann, Tobias Benthaus, Thomas Büchner, Stephanie Schneider, Bernhard Wörmann, Eva Hoster, Jan Braess, Annika Dufour, Wolfgang E. Berdel, Maria-Cristina Sauerland, Evelyn Zellmeier, Stefan K. Bohlander, Friederike Schneider, Karsten Spiekermann, and Klaus H. Metzeler
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Adult ,Male ,Cancer Research ,NPM1 ,Myeloid ,Adolescent ,medicine.disease_cause ,Young Adult ,CEBPA ,Biomarkers, Tumor ,medicine ,Humans ,Survival rate ,Alleles ,Aged ,Neoplasm Staging ,Oligonucleotide Array Sequence Analysis ,Aged, 80 and over ,Mutation ,Gene Expression Regulation, Leukemic ,business.industry ,Gene Expression Profiling ,Nuclear Proteins ,Myeloid leukemia ,MINIMAL RESIDUAL DISEASE ,BINDING-PROTEIN-ALPHA ,C/EBP-ALPHA ,PROGNOSTIC-SIGNIFICANCE ,EXPRESSION PROFILE ,TANDEM DUPLICATION ,RELAPSE SAMPLES ,AML ,FLT3 ,CYTOGENETICS ,Histone-Lysine N-Methyltransferase ,Middle Aged ,Prognosis ,medicine.disease ,Survival Rate ,Gene expression profiling ,Leukemia, Myeloid, Acute ,Leukemia ,Treatment Outcome ,medicine.anatomical_structure ,fms-Like Tyrosine Kinase 3 ,Oncology ,Karyotyping ,CCAAT-Enhancer-Binding Proteins ,Cancer research ,Female ,business ,Nucleophosmin ,Myeloid-Lymphoid Leukemia Protein - Abstract
Purpose CEBPA mutations are found as either biallelic (biCEBPA) or monoallelic (moCEBPA). We set out to explore whether the kind of CEBPA mutation is of prognostic relevance in cytogenetically normal (CN) acute myeloid leukemia (AML). Patients and Methods Four hundred sixty-seven homogeneously treated patients with CN-AML were subdivided into moCEBPA, biCEBPA, and wild-type (wt) CEBPA patients. The subgroups were analyzed for clinical parameters and for additional mutations in the NPM1, FLT3, and MLL genes. Furthermore, we obtained gene expression profiles using oligonucleotide microarrays. Results Only patients with biCEBPA had an improved median overall survival when compared with patients with wtCEBPA (not reached v 20.4 months, respectively; P = .018), whereas patients with moCEBPA (20.9 months) and wtCEBPA had a similar outcome (P = .506). Multivariable analysis confirmed biCEBPA, but not moCEBPA, mutations as an independent favorable prognostic factor. Interestingly, biCEBPA mutations, compared with wtCEBPA, were never associated with mutated NPM1 (0% v 43%, respectively; P < .001) and rarely associated with FLT3 internal tandem duplication (ITD; 5% v 23%, respectively; P = .059), whereas patients with moCEBPA had a similar frequency of mutated NPM1 and a significantly higher association with FLT3-ITD compared with patients with wtCEBPA (44% v 23%, respectively; P = .037). Furthermore, patients with biCEBPA showed a homogeneous gene expression profile that was characterized by downregulation of HOX genes, whereas patients with moCEBPA showed greater heterogeneity in their gene expression profiles. Conclusion Biallelic disruption of the N and C terminus of CEBPA is required for the favorable clinical outcome of CEBPA-mutated patients and represents a distinct molecular subtype of CN-AML with a different frequency of associated gene mutations. These findings are of great significance for risk-adapted therapeutic strategies in AML.
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- 2010
17. Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis
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Alexander Graf, Max Hubmann, Ulrich Mansmann, Stefan Krebs, Zlatana Pasalic, Bianka Ksienzyk, Annika Dufour, Tobias Herold, Wolfgang E. Berdel, Bernhard J. Woermann, Gerhard Ehninger, Vindi Jurinovic, Martin Bornhäuser, Friedrich Stölzel, Maria Cristina Sauerland, Philipp A. Greif, Sebastian Vosberg, Luise Hartmann, Helmut Blum, C. Röllig, Stefan K. Bohlander, Stephanie Schneider, Klaus H. Metzeler, Purvi M. Kakadia, Karsten Spiekermann, Evelyn Zellmeier, Thomas Büchner, and Wolfgang Hiddemann
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Adult ,Male ,Poor prognosis ,Spliceosome ,Adolescent ,Immunology ,Trisomy ,Biology ,Bioinformatics ,Biochemistry ,Disease-Free Survival ,hemic and lymphatic diseases ,Germany ,medicine ,Humans ,neoplasms ,Gene ,Exome sequencing ,Aged ,Aged, 80 and over ,Chromosomes, Human, Pair 13 ,Gene Expression Regulation, Leukemic ,Clinical course ,Myeloid leukemia ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Neoplasm Proteins ,Up-Regulation ,Survival Rate ,Leukemia, Myeloid, Acute ,Homogeneous ,Cancer research ,Female - Abstract
Isolated trisomy 13 (AML+13) is a rare chromosomal abnormality in acute myeloid leukemia (AML), and its prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and studied their biological characteristics by exome sequencing, targeted sequencing of candidate genes and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, p=0.006; median OS 9.3 vs. 14.8 months, p=0.004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%, of AML+13 patients. Moreover, recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogenous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogenous leukemia subgroup with alterations in a few critical cellular pathways. These studies were registered at clinicaltrials.gov, identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.
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- 2014
18. The NPM1 mutation type has no impact on survival in cytogenetically normal AML
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Bianka Ksienzyk, Jan Braess, Stephanie Schneider, Wolfgang E. Berdel, Thomas Buechner, Wolfgang Hiddemann, Evelyn Zellmeier, Friederike Pastore, Cristina Sauerland, Utz Krug, Philipp A. Greif, Bernhard J. Woermann, Karsten Spiekermann, Achim Heinecke, and Gudrun Mellert
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Oncology ,Male ,Myeloid ,Epidemiology ,lcsh:Medicine ,Gene Expression ,medicine.disease_cause ,Bioinformatics ,Hematologic Cancers and Related Disorders ,Risk Factors ,hemic and lymphatic diseases ,Medicine and Health Sciences ,lcsh:Science ,Mutation ,Multidisciplinary ,Remission Induction ,Cytarabine ,Myeloid leukemia ,Nuclear Proteins ,Induction Chemotherapy ,Hematology ,Middle Aged ,Prognosis ,Myeloid Leukemia ,Leukemia ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Treatment Outcome ,Female ,Nucleophosmin ,Cancer Epidemiology ,Research Article ,Adult ,Acute Myeloid Leukemia ,medicine.medical_specialty ,NPM1 ,Antineoplastic Agents ,Frameshift mutation ,Molecular Genetics ,Internal medicine ,Leukemias ,medicine ,Genetics ,Humans ,Thioguanine ,Survival analysis ,Aged ,business.industry ,lcsh:R ,Daunorubicin ,Cytogenetics ,Biology and Life Sciences ,medicine.disease ,Survival Analysis ,fms-Like Tyrosine Kinase 3 ,Karyotyping ,lcsh:Q ,Mitoxantrone ,business - Abstract
NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML.
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- 2014
19. Molecular response assessment by quantitativerael-time polymerase chain reaction after induction therapy in NPM1-mutated patients identifies patients at high risk for relapse
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Marion Subklewe, Thomas Büchner, Michael Fiegl, Wolfgang E. Berdel, Wolfgang Hiddemann, Annika Dufour, Max Hubmann, Karsten Spiekermann, Maria-Cristina Sauerland, Stephanie Schneider, Thomas Köhnke, Jan Braess, Evelyn Zellmeier, Bernhard Wörmann, Stefan K. Bohlander, and Eva Hoster
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Adult ,Male ,Oncology ,medicine.medical_specialty ,Adolescent ,Real-Time Polymerase Chain Reaction ,Acute Myeloid Leukemia ,Mrd ,Minimal Residual Disease ,Npm1 ,Rt-pcr ,Young Adult ,Recurrence ,Risk Factors ,Internal medicine ,medicine ,Humans ,Cumulative incidence ,Prospective Studies ,Prospective cohort study ,Aged ,Retrospective Studies ,Aged, 80 and over ,business.industry ,Hazard ratio ,Nuclear Proteins ,Myeloid leukemia ,Retrospective cohort study ,Induction Chemotherapy ,Articles ,Hematology ,Middle Aged ,medicine.disease ,Minimal residual disease ,Surgery ,Survival Rate ,Clinical trial ,Leukemia, Myeloid, Acute ,Leukemia ,Treatment Outcome ,Mutation ,Female ,business ,Nucleophosmin ,Follow-Up Studies - Abstract
Monitoring minimal residual disease is an important way to identify patients with acute myeloid leukemia at high risk of relapse. In this study we investigated the prognostic potential of minimal residual disease monitoring by quantitative real-time polymerase chain reaction analysis of NPM1 mutations in patients treated in the AMLCG 1999, 2004 and 2008 trials. Minimal residual disease was monitored - in aplasia, after induction therapy, after consolidation therapy, and during follow-up - in 588 samples from 158 patients positive for NPM1 mutations A, B and D (with a sensitivity of 10(-6)). One hundred and twenty-seven patients (80.4%) achieved complete remission after induction therapy and, of these, 56 patients (44.1%) relapsed. At each checkpoint, minimal residual disease cut-offs were calculated. After induction therapy a cut-off NPM1 mutation ratio of 0.01 was associated with a high hazard ratio of 4.26 and the highest sensitivity of 76% for the prediction of relapse. This was reflected in a cumulative incidence of relapse after 2 years of 77.8% for patients with ratios above the cut-off versus 26.4% for those with ratios below the cut-off. In the favorable subgroup according to European LeukemiaNet, the cut-off after induction therapy also separated the cohort into two prognostic groups with a cumulative incidence of relapse of 76% versus 6% after 2 years. Our data demonstrate that in addition to pre-therapeutic factors, the course of minimal residual disease in an individual is an important prognostic factor and could be included in clinical trials for the guidance of post-remission therapy. The trials from which data were obtained were registered at www.clinicaltrials.gov (#NCT01382147, #NCT00266136) and at the European Leukemia Trial Registry (#LN_AMLINT2004_230).
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- 2014
20. Combined molecular and clinical prognostic index for relapse and survival in cytogenetically normal acute myeloid leukemia
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Annika Dufour, Gudrun Mellert, Guido Marcucci, Eva Hoster, Kati Maharry, Bernhard J. Woermann, Wolfgang E. Berdel, Stefan K. Bohlander, Maria Cristina Sauerland, Michaela Feuring-Buske, Bianka Ksienzyk, Utz Krug, Stephanie Schneider, Evelyn Zellmeier, Christian Buske, Clara D. Bloomfield, Purvi M. Kakadia, Michael Unterhalt, Tobias Benthaus, Klaus H. Metzeler, Karsten Spiekermann, Achim Heinecke, Wolfgang Hiddemann, Thomas Buechner, Jan Braess, and Friederike Pastore
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Oncology ,Male ,Cancer Research ,Myeloid ,Time Factors ,DNA Mutational Analysis ,Kaplan-Meier Estimate ,Recurrence ,Risk Factors ,Germany ,CEBPA ,Medicine ,Aged, 80 and over ,Age Factors ,Myeloid leukemia ,Nuclear Proteins ,ORIGINAL REPORTS ,Middle Aged ,Leukemia ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Phenotype ,Treatment Outcome ,Cytogenetic Analysis ,Female ,Nucleophosmin ,Adult ,medicine.medical_specialty ,NPM1 ,Adolescent ,Risk Assessment ,Disease-Free Survival ,Decision Support Techniques ,Young Adult ,Predictive Value of Tests ,Internal medicine ,Humans ,Genetic Predisposition to Disease ,Aged ,Proportional Hazards Models ,Performance status ,business.industry ,Proportional hazards model ,Cancer ,Reproducibility of Results ,medicine.disease ,fms-Like Tyrosine Kinase 3 ,Immunology ,Mutation ,CCAAT-Enhancer-Binding Proteins ,business - Abstract
Purpose Cytogenetically normal (CN) acute myeloid leukemia (AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician, it is difficult to summarize the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pretreatment patient and disease characteristics. Patients and Methods Two prognostic indices for CN-AML (PINA), one regarding overall survival (OS; PINAOS) and the other regarding relapse-free survival (RFS; PINARFS), were derived from data of 572 patients with CN-AML treated within the AML Cooperative Group 99 study ( www.aml-score.org ). Results On the basis of age (median, 60 years; range, 17 to 85 years), performance status, WBC count, and mutation status of NPM1, CEBPA, and FLT3-internal tandem duplication, patients were classified into the following three risk groups according to PINAOS and PINARFS: 29% of all patients and 32% of 381 responding patients had low-risk disease (5-year OS, 74%; 5-year RFS, 55%); 56% of all patients and 39% of responding patients had intermediate-risk disease (5-year OS, 28%; 5-year RFS, 27%), and 15% of all patients and 29% of responding patients had high-risk disease (5-year OS, 3%; 5-year RFS, 5%), respectively. PINAOS and PINARFS stratified outcome within European LeukemiaNet genetic groups. Both indices were confirmed on independent data from Cancer and Leukemia Group B/Alliance trials. Conclusion We have developed and validated, to our knowledge, the first prognostic indices specifically designed for adult patients of all ages with CN-AML that combine well-established molecular and clinical variables and that are easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care through risk-adapted therapy of CN-AML.
- Published
- 2014
21. Evolutionary Patterns of Cytogenetically Normal Acute Myeloid Leukemia Correlate with Time to Relapse
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Alexander Graf, S. Schneider, Stefan K. Bohlander, Nikola P. Konstandin, Stephan Wolf, Daniela Schumacher, Helmut Blum, Evelyn Zellmeier, Sebastian Vosberg, Wolfgang Hiddemann, Bianka Ksienzyk, Claudia D. Baldus, Stefan Krebs, Friederike Pastore, Luise Hartmann, Martin Neumann, Kathrin Bräundl, Philipp A. Greif, Klaus H. Metzeler, and Karsten Spiekermann
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Oncology ,medicine.medical_specialty ,Chemotherapy ,IDH1 ,business.industry ,medicine.medical_treatment ,Immunology ,Clone (cell biology) ,Induction chemotherapy ,Salvage therapy ,Cell Biology ,Hematology ,Biochemistry ,Somatic evolution in cancer ,Log-rank test ,Germline mutation ,Internal medicine ,medicine ,business - Abstract
Even though two-thirds of acute myeloid leukemia (AML) patients respond to induction chemotherapy and achieve complete remission (CR), the majority of these patients will eventually relapse. The time from CR to relapse is an important clinical indicator of disease aggressiveness, as patients relapsing within the first 6 months after initial diagnosis have a poorer prognosis in terms of response to salvage therapy and overall survival compared to patients with a later relapse. To learn about the evolution during the course of disease, we analyzed the somatic mutation patterns from initial diagnosis to relapse in 50 cytogenetically normal (CN) AML patients. Based on the ELN classification, 38% of the patients (n=19) were assigned as "favorable" at diagnosis, all other patients were classified as "intermediate-I". ELN classification was associated with time to relapse as "intermediate-I" patients relapsed earlier than "favorable" patients (median 9.3 months vs. 16.1 months, p=0.008, log-rank test). Somatic alterations were detected by exome sequencing and confirmed by targeted amplicon sequencing of matched diagnostic, remission and relapse samples. FLT3-ITD and NPM1 mutation status were obtained from routine diagnostic tests as the reliable detection of these markers by NGS remains challenging. The vast majority of somatic alterations were present both at diagnosis and at relapse, hereafter referred to as stable mutations (70%, Fig. 1A). All patients in our cohort had ≥1 stable mutation with DNMT3A being the most stably altered gene. In 47 out of 50 patients (94%), we observed mutations that were only found at diagnosis or only at relapse. Based on the mutation patterns, four distinct 'evolutionary' subgroups of patients were defined (Fig. 1B): (I) patients with an identical mutation profile at diagnosis and at relapse ("stable", n=3, 6%), (II) patients who gained mutations at relapse ("stable + gain", n=24, 48%), (III) patients that lost mutations at relapse ("stable + loss", n=8, 16%), and (IV) patients with both loss and gain of mutations at relapse ("mixed", n=15, 30%). Mutations that were lost during the course of the disease were detected in e.g. PTPN11 or NRAS. Relapse-specific mutations were identified in e.g. IDH1/2, WT1, KPNB1 or KDM6A. Evolutionary subgroups showed differences in time to relapse (Fig. 1C). Patients with "stable + loss" relapsed earlier (median 4.1 months) than patients with gain of mutation at relapse (groups "stable + gain" and "mixed", median 12.2 months). All patients in the category "stable + loss" developed relapse within the first year after complete remission. The "stable" group of 3 patients showed an intermediate time to relapse (median 9.6 months), but was too small for a statistically valid comparison. Ultimately, the genetic evolution of CN-AML patients without gain of new mutations at relapse (categories "stable" and "stable + loss") was associated with significantly earlier relapse compared to patients that gained mutations at relapse (categories "stable + gain" and "mixed", Fig. 1D, p=0.001, log-rank test). Distinct predominant patterns of clonal evolution were observed in the ELN genetic groups, as only one patient of the "stable + loss" group was initially classified as "favorable". Interestingly, applying the ELN classification on relapse samples revealed a switch from "favorable" to "intermediate-I" in six patients, all with gain of mutations at relapse. This points towards more aggressive genetic profiles at relapse in these patients. The acquisition of mutations and/or the outgrowth of a resistant clone during/after chemotherapy might require a longer time or is per se associated with a longer time to relapse and a more favorable prognosis. Loss of mutations at relapse suggest the presence of two clones at diagnosis, with a chemotherapy resistant clone expanding after the eradication of a chemotherapy sensitive clone. As both clones share mutations and only the sensitive clone contains specific alterations, the resistant clone might be an ancestor of the sensitive clone. Taken together, in some patients the AML cells may require additional genetic alterations to become chemotherapy resistant, whereas in other patients the selective eradication of a sensitive clone is a potential mechanism underlying disease progression. Understanding the evolution of AML under selective pressure of chemotherapy is essential to cure or prevent AML relapse. Disclosures Hiddemann: Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.
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- 2016
22. Comparison of FLT3-ITD Detection By High-Throughput Amplicon Sequencing to Routine Diagnostics - a Retrospective Analysis of AMLCG Study Patients
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Christina Sauerland, Evelyn Zellmeier, Karsten Spiekermann, Annika Dufour, Bianka Ksienzyk, Stephanie Schneider, Stefan K. Bohlander, Wolfgang E. Berdel, Kathrin Bräundl, Max Hubmann, Jan Braess, Philipp A. Greif, Wolfgang Hiddemann, Bernhard Wörmann, Egor Harin, Katrin Reiter, Sebastian Vosberg, and Thomas Büchner
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Oncology ,medicine.medical_specialty ,biology ,business.industry ,Immunology ,Cell Biology ,Hematology ,Amplicon ,Biochemistry ,Relapse free survival ,body regions ,KMT2A ,Internal medicine ,Induction therapy ,CEBPA ,biology.protein ,Retrospective analysis ,Amplicon sequencing ,medicine ,business ,psychological phenomena and processes ,Flt3 itd - Abstract
Introduction In Acute Myeloid Leukemia (AML) internal tandem duplications (ITD) in the fms-related tyrosine kinase 3 (FLT3) are a frequent event associated with an unfavorable prognosis. At diagnosis, the FLT3-ITD status is routinely assessed by fragment analysis of PCR-amplified cDNA. However, this assay only provides information on the length but not on the position and sequence of the insertion. Therefore, it is attractive to overcome this limitation by the use of high-throughput amplicon sequencing (HTAS) as an alternative strategy for FLT3-ITD detection. To proof the feasibility and accuracy of this approach we performed HTAS on 260 AML patients, of which 250 were FLT3-ITD positive according to routine diagnostics. Patients and Methods All samples were obtained from patients treated on the German Acute Myeloid Leukemia Cooperative Group (AMLCG) trials 1999, 2004 and 2008. All patients received intensive induction therapy with curative intent. At diagnosis, patients had a median age of 60 years (range, 18-80 years). Additional molecular marker were screened routinely: NPM1 mutation (62%), KMT2A-PTD (8%), CEBPA mutations (8%). According to the ELN-classification patients clustered into the following groups: ELN intermediate I (70%), intermediate II (21%) or adverse (9%). A normal karyotype was observed in 71%, while 29% had an aberrant karyotpye. All patient samples were analyzed routinely by FLT3 fragment analysis. For HTAS of FLT3 we used custom FLT3 cDNA primers including barcode and adapter-sequences enabling a one-step PCR-protocol. Sequencing was performed (2x250bp paired end) on a MiSeq (Illumina). Per run, up to 96 samples were sequenced, yielding a median of 79,110 reads per amplicon (range: 31,996 - 259,783). As controls, cDNA from FLT3-ITD positive (Molm-13) and negative (HL60) cell lines were used. Sequencing data were aligned to the FLT3 cDNA reference (NM_004119.2) and FLT3-ITDs were called using Pindel software (version 0.2.5a7). Results Based on the HTAS results obtained from the FLT3 wild-type control HL60, we set the cut-off for ITD detection at a variant allele frequency (VAF) of 0.5%. Using this threshold, all patients which were assessed to be FLT3-ITD negative in diagnostic routine, were also negative in HTAS. FLT3-ITDs were detected by HTAS in 242 out of 250 (97%) patients who were FLT3-ITD positive patients according to routine fragment analysis. For six out of the 8 remaining patients, no valid ITD was detected by HTAS possibly due to one of the following reasons: a low mutational burden resulting in a VAF below the cut-off level (n=4) or a deletion near the ITD (n=2) potentially interfering with ITD-detection. In total, 308 ITDs were detected in 242 patients by HTAS while in these patients 282 ITDs were detected by routine diagnostics. Of note, HTAS missed 13 subclonal ITDs reported in routine, while 39 additional subclonal ITDs were detected by HTAS only. Overall, HTAS detected a higher number of ITDs per patient (mean: 1.27; range: 1-4) compared to fragment analysis (mean: 1.17; range: 1-3). Patients with more than one ITD according to HTAS showed a trend towards shorter overall and relapse free survival (p=0.105, p=0.104 respectively; Figure 1A). The ITD position (i.e. affected FLT3 domain) based on HTAS did not impact on clinical outcome. There was a significant correlation between the FLT3-ITD levels detected by fragment analysis and HTAS (Pearson, R=0.801, p=0.01; Figure 1B). High FLT3-ITD levels measured by fragment analysis had a significant impact on RFS, whereas this effect was not observed for FLT3-ITD levels measured by HTAS. The quantification of FLT3-ITD by HTAS might be hampered by underestimation of the VAFs of long ITDs that are less likely to be mapped correctly compared to shorter ITDs. Conclusion In summary, our study demonstrates the feasibility of HTAS for FLT3-ITD detection in AML. In particular, the identification of subclonal ITDs with high sensitivity provides additional information with potential prognostic value. Thus, HTAS may serve as a robust tool that could be implemented in future diagnostic routines. However, bioinformatic algorithms for ITD detection may need further improvement, e.g. to optimize ITD quantification and to facilitate the detection of ITDs in combination with deletions. Disclosures Hiddemann: Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.
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- 2016
23. Exome sequencing identifies recurring FLT3 N676K mutations in core-binding factor leukemia
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Bianka Ksienzyk, Sabrina Opatz, Helmut Blum, Tobias Herold, Philipp A. Greif, Nikola P. Konstandin, Annika Dufour, Sebastian Vosberg, Maria Cristina Sauerland, Wolfgang E. Berdel, Wolfgang Hiddemann, Thomas Büchner, Jan Braess, Bernhard J. Woermann, Evelyn Zellmeier, Karl-Peter Hopfner, Stefan Krebs, Harald Polzer, Stephanie Schneider, Alexander Graf, Karsten Spiekermann, Purvi M. Kakadia, and Stefan K. Bohlander
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Adult ,Male ,Models, Molecular ,Adolescent ,Oncogene Proteins, Fusion ,Immunology ,DNA Mutational Analysis ,Molecular Sequence Data ,Apoptosis ,Biology ,medicine.disease_cause ,Biochemistry ,Core Binding Factor beta Subunit ,Fusion gene ,chemistry.chemical_compound ,hemic and lymphatic diseases ,medicine ,MYH11 ,Humans ,Exome ,Benzothiazoles ,Protein Kinase Inhibitors ,Exome sequencing ,Cell Proliferation ,Genetics ,Gene Rearrangement ,Mutation ,Leukemia ,Base Sequence ,Gene Expression Regulation, Leukemic ,Phenylurea Compounds ,Myeloid leukemia ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Staurosporine ,Cell Transformation, Neoplastic ,RUNX1 ,chemistry ,Amino Acid Substitution ,fms-Like Tyrosine Kinase 3 ,embryonic structures ,Cancer research ,Cytokines ,Female ,Tyrosine kinase - Abstract
The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB / MYH11 or RUNX1 / RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 ( FLT3 ) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB / MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias. This trial was registered at www.clinicaltrials.gov as #NCT00266136.
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- 2013
24. GATA2 zinc finger 1 mutations associated with biallelic CEBPA mutations define a unique genetic entity of acute myeloid leukemia
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Friederike Schneider, Jan Braess, Andreas Hauser, Helmut Blum, Martin Stanulla, Tobias Herold, Jutta Sturm, Eva Hoster, Wolfgang Hiddemann, Purvi M. Kakadia, Nikola P. Konstandin, Wolfgang E. Berdel, Belay Tizazu, Tobias Benthaus, Thomas Büchner, Evelyn Zellmeier, Stefan K. Bohlander, Karl-Peter Hopfner, Maria Cristina Sauerland, Stefan Krebs, Petra Dörge, Bianka Ksienzyk, Annika Dufour, Alexander Graf, Stephanie Schneider, Bernhard J. Woermann, Karsten Spiekermann, Philipp A. Greif, and Marjan Yaghmaie
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Adult ,Models, Molecular ,Transcriptional Activation ,Immunology ,DNA Mutational Analysis ,Karyotype ,Molecular Sequence Data ,Biology ,Biochemistry ,Gene Frequency ,CEBPA ,medicine ,Humans ,Exome ,Amino Acid Sequence ,Gene ,Exome sequencing ,Alleles ,Genetics ,Zinc finger ,Base Sequence ,GATA2 ,Myeloid leukemia ,Zinc Fingers ,Cell Biology ,Hematology ,DNA, Neoplasm ,medicine.disease ,Prognosis ,GATA2 Transcription Factor ,Leukemia ,Leukemia, Myeloid, Acute ,Cytogenetic Analysis ,Mutation ,Cancer research ,CCAAT-Enhancer-Binding Proteins - Abstract
Cytogenetically normal acute myeloid leukemia (CN-AML) with biallelic CEBPA gene mutations (biCEPBA) represents a distinct disease entity with a favorable clinical outcome. So far, it is not known whether other genetic alterations cooperate with biCEBPA mutations during leukemogenesis. To identify additional mutations, we performed whole exome sequencing of 5 biCEBPA patients and detected somatic GATA2 zinc finger 1 (ZF1) mutations in 2 of 5 cases. Both GATA2 and CEBPA are transcription factors crucial for hematopoietic development. Inherited or acquired mutations in both genes have been associated with leukemogenesis. Further mutational screening detected novel GATA2 ZF1 mutations in 13 of 33 biCEBPA-positive CN-AML patients (13/33, 39.4%). No GATA2 mutations were found in 38 CN-AML patients with a monoallelic CEBPA mutation and in 89 CN-AML patients with wild-type CEBPA status. The presence of additional GATA2 mutations (n=10) did not significantly influence the clinical outcome of 26 biCEBPA-positive patients. In reporter gene assays, all tested GATA2 ZF1 mutants showed reduced capacity to enhance CEBPA-mediated activation of transcription, suggesting that the GATA2 ZF1 mutations may collaborate with biCEPBA mutations to deregulate target genes during malignant transformation. We thus provide evidence for a genetically distinct subgroup of CN-AML. The German AML cooperative group trials 1999 and 2008 are registered with the identifiers NCT00266136 and NCT01382147 at www.clinicaltrials.gov.
- Published
- 2012
25. The FLT3ITD mRNA level has a high prognostic impact in NPM1 mutated, but not in NPM1 unmutated, AML with a normal karyotype
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Gudrun Mellert, Annika Dufour, Michaela Feuring-Buske, Stefan K. Bohlander, Karsten Spiekermann, Friederike Schneider, Maria Cristina Sauerland, Achim Heinecke, Wolfgang E. Berdel, Michael Unterhalt, Jan Braess, Evelyn Zellmeier, Bernhard Wörmann, Stephanie Schneider, Tobias Benthaus, Thomas Büchner, Wolfgang Hiddemann, Eva Hoster, Purvi M. Kakadia, and Christian Buske
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Oncology ,Adult ,Male ,medicine.medical_specialty ,NPM1 ,Myeloid ,Immunology ,Karyotype ,Biology ,Biochemistry ,Internal medicine ,medicine ,Humans ,RNA, Messenger ,Survival analysis ,Aged ,Aged, 80 and over ,Proportional hazards model ,Hazard ratio ,Myeloid leukemia ,Nuclear Proteins ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Prognosis ,Combined Modality Therapy ,Survival Analysis ,Confidence interval ,Leukemia ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Treatment Outcome ,fms-Like Tyrosine Kinase 3 ,Tandem Repeat Sequences ,Mutation ,Female ,Nucleophosmin - Abstract
The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level—measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3 + 1)—on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML.
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- 2012
26. Mutations of Genes Linked to Epigenetic Regulation Are Frequently Gained in Relapsed Cytogenetically Normal Acute Myeloid Leukemia
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Nikola P. Konstandin, Wolfgang Hiddemann, Alexander Graf, Evelyn Zellmeier, Friederike Pastore, Stefan Krebs, Bianka Ksienzyk, Stephanie Schneider, Klaus H. Metzeler, Helmut Blum, Sebastian Vosberg, Martin Neumann, Kathrin Bräundl, Karsten Spiekermann, Daniela Schumacher, Stefan K. Bohlander, Claudia D. Baldus, Philipp A. Greif, and Luise Hartmann
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Mutation ,NPM1 ,Myeloid ,IDH1 ,Immunology ,Cell Biology ,Hematology ,Biology ,Bioinformatics ,medicine.disease_cause ,medicine.disease ,Biochemistry ,IDH2 ,Germline ,Leukemia ,medicine.anatomical_structure ,medicine ,Cancer research ,Epigenetics - Abstract
Cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous disease with regard to genetic alterations and clinical outcome. Recent sequencing studies categorized the growing number of recurrently mutated genes into different functional groups, e.g. myeloid transcription factors, tumor suppressors, signal transducers, chromatin modifiers, cohesin-complex and spliceosome-complex. We set out to characterize mutations in genes linked to epigenetic regulation during the progression of CN-AML. Besides genes directly involved in chromatin modification (i.e. DNMT3A, TET2, MLL, ASXL1, KDM6A, KDM2A, NSD1 and EZH2), we also studied mutations in WT1 and IDH1/2 since they are known to inhibit TET2 function. Targeted sequencing of 46 genes related to leukemia (mean coverage >500x) was performed on matched diagnostic, remission and relapse samples of 50 patients with CN-AML (median age: 66, range: 21-89). We called somatic variants at diagnosis or at relapse and filtered for mutations with translational consequences, excluding known error-prone genes and common germline polymorphisms (dbSNP 138; MAF>=1%). At diagnosis, 36/50 patients (72%) carried a total of 48 mutations in epigenetic regulators (Figure 1). The majority of patients harbored a single mutation affecting this functional group, while 2 or 3 mutations were observed in 9 and 1 patient(s), respectively. The median variant allele frequency (VAF) of the mutations was 42% (range: 22-98%), indicating that mutations in epigenetic regulators are early events and are present in the founding clone. Of the 48 mutations detected at diagnosis, only 2 were lost at relapse, highlighting the stability of these lesions during disease progression. Moreover, in 12/50 patients (24%), mutations in epigenetic regulators were acquired at relapse. All but one of these patients already had a mutation in another epigenetic regulator at diagnosis. We did not identify patients who acquired DNMT3A, TET2 or ASXL1 mutations during disease progression. However, mutations in WT1, IDH1, and KDM6A were gained in several patients at relapse. In 4/13 cases, the gained mutations were already detectable at low levels at diagnosis (median VAF: 2.9%, range: 0.3-6%, mean coverage at the investigated sites: 629x, range: 85-1625x). We also evaluated the presence of these mutations in remission: In 18 out of 36 (50%) patients, some of the mutations affecting DNMT3A (n=14), TET2 (n=3) or IDH2 (n=2) were present at a VAF >5% (median: 22%, range: 9-75%) in cytomorphologically defined complete remission, suggesting the persistence of pre-leukemic clones with limited response to chemotherapy. Longer relapse-free survival was observed in patients with DNMT3A mutations that did not persist at remission (np-DNMT3A) in comparison to patients with persisting DNMT3A mutations (p-DNMT3A). Remarkably, the latter group was enriched for patients that also harbored FLT3 internal tandem duplications (ITDs) (10/14 versus 1/8; Fisher's exact test, p=0.02). The vast majority of p-DNMT3A showed alterations of R882, whereas mutations at other positions of DNMT3A tended to be undetectable in remission. When including the NPM1 status, only 1/8 patient with np-DNMT3A was triple mutated, compared to 11/14 patients with p-DNMT3A, suggesting that co-occurrence of DNMT3A, FLT3- ITD and NPM1 c is associated with p-DNMT3A (p=0.006). In summary, we show that a high proportion of patients (72%) with relapsing CN-AML is affected by mutations in genes linked to epigenetic regulation. The stability of these mutations between diagnosis and relapse in combination with their acquisition during disease progression, as well as the frequent persistence of DNMT3A, TET2 and IDH2 mutations during remission underscore the necessity for new therapeutic approaches. The striking association of DNMT3A R882 mutations with NPM1 c and FLT3 -ITD suggest a unique mechanism of oncogenic collaboration. Persistence of DNMT3A R882 mutations may indicate a fertile ground for relapse. Further studies will be required to clarify whether the actual relapse arises from a preleukemic clone harboring only the founder mutation or from residual leukemia cells containing several genetic lesions. Disclosures No relevant conflicts of interest to declare.
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- 2015
27. DNMT3A Mutations Associate with Shorter Survival and Modulate the Prognostic Impact of Mutated NPM1: an Analysis Based on Comprehensive Mutational Screening of 660 AML Patients Treated on German AML Cooperative Group (AMLCG) Trials
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Luise Hartmann, Marion Subklewe, Susanne Amler, Andreas Faldum, Annika Dufour, Cristina Sauerland, Michael Fiegl, Tobias Herold, Jan Braess, Kathrin Bräundl, Wolfgang E. Berdel, Evelyn Zellmeier, Bianka Ksienzyk, Stefan K. Bohlander, Klaus H. Metzeler, Bernhard Wörmann, Nikola P. Konstandin, Karsten Spiekermann, Stephanie Schneider, Thomas Büchner, Philipp A. Greif, Utz Krug, Maja Rothenberg-Thurley, and Wolfgang Hiddemann
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Oncology ,medicine.medical_specialty ,NPM1 ,Myeloid ,Immunology ,Hazard ratio ,Cytogenetics ,Induction chemotherapy ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,Bioinformatics ,Biochemistry ,medicine.anatomical_structure ,Internal medicine ,Genotype ,medicine ,Missense mutation - Abstract
Background: Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia (AML), affecting ~20% of patients (pts) and 30-40% of those with cytogenetically normal (CN-) AML. Although several groups have investigated their prognostic relevance, most studies focused on younger adults (=60 y) AML pts treated on German AML Cooperative Group (AMLCG) protocols, and studied the association between DNMT3A mutations and outcomes. Patients and Methods: We analyzed pretreatment blood or bone marrow specimens from 660 adult AML pts who received intensive induction chemotherapy on two consecutive phase III trials (AMLCG-1999, n=388, and AMLCG-2008, n=272; median age, 57y, range, 18-86y). Sequence variants in DNMT3A exons 7-23 and other genes known to be mutated in myeloid neoplasms were analyzed by multiplexed amplicon resequencing (Agilent Haloplex). Sequencing was performed on an Illumina MiSeq instrument using 2x250bp paired-end reads. Variants were classified as known/putative driver mutations, variants of unknown significance, or known germline polymorphisms based on published data including dbSNP, the Catalogue Of Somatic Mutations In Cancer (COSMIC) and The Cancer Genome Atlas (TCGA). Cytogenetic analyses were performed centrally. Results: We identified 223 DNMT3A mutations in 207/660 pts (31%), including 180/449 pts (40%) with intermediate-risk cytogenetics according to the MRC classification (P 1 type of DNMT3A mutation. DNMT3A mutations tended to be more frequent in older compared to younger pts (35% vs. 28%, P =.08) and were associated with female sex (38% vs 26% in males; P Conclusion: In our cohort of intensively treated AML pts covering a broad age range, we found that DNMT3A mutations associate with inferior survival and modulate the prognostic impact of mutated NPM1, confirming data recently reported by the MRC group (Gale et al., J Clin Oncol 33:2072). In contrast to this and other published reports, we observed no outcome differences between different types of DNMT3A mutations. Information on DNMT3A mutation status further refined the risk stratification of CN-AML based on the NPM1 mutated / FLT3-ITD negative genotype, supporting a role for DNMT3A mutations as a prognostic marker. Figure 1. Figure 1. Disclosures Subklewe: AMGEN Research (Munich): Research Funding. Krug:Boehringer Ingelheim: Research Funding; Novartis; BMS; Roche; Boehringer Ingelheim; Bayer: Honoraria; Sunesis: Speakers Bureau; Sunesis; Clavis Pharma; usa Pharma, Catapult Cell Therapy, Gilead, Roche: Membership on an entity's Board of Directors or advisory committees.
- Published
- 2015
28. Monitoring minimal residual disease in acute myeloid leukaemia with NPM1 mutations by quantitative PCR: clonal evolution is a limiting factor
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Wolfgang Hiddemann, Marlene Seibl, Stephanie Schneider, Annika Dufour, Karsten Spiekermann, Gudrun Mellert, Stefan K. Bohlander, Christina Papadaki, and Evelyn Zellmeier
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Adult ,Genetic Markers ,NPM1 ,Myeloid ,Neoplasm, Residual ,Biology ,Somatic evolution in cancer ,Sensitivity and Specificity ,law.invention ,Young Adult ,law ,Recurrence ,hemic and lymphatic diseases ,medicine ,Humans ,Polymerase chain reaction ,Aged ,Reverse Transcriptase Polymerase Chain Reaction ,Nuclear Proteins ,Hematology ,Middle Aged ,Molecular biology ,Minimal residual disease ,Housekeeping gene ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Real-time polymerase chain reaction ,Mutation (genetic algorithm) ,Mutation ,Cancer research ,Neoplastic Stem Cells ,Nucleophosmin - Abstract
Nucleophosmin (NPM1) mutations in exon 12 represent the most frequent molecular aberrations in adult patients with acute myeloid leukaemia (AML). Molecular detection of NPM1 mutation A could be a useful marker for routine monitoring of minimal residual disease (MRD). We established a calibrator-normalized relative quantification real-time polymerase chain reaction (PCR) assay for NPM1 mutation A. ABL1 was used as a reference housekeeping gene and the NPM1 mutation A-containing OCI/AML3 cell line as a calibrator. Relative quantification was performed by calculating the NPM1 mutation A/ABL1 ratio which was normalized to the NPM1 mutation A/ABL1 ratio of OCI/AML3 calibrator cDNA. The assay showed a sensitivity of 10(-5). The clinical usefulness was evaluated by monitoring MRD in 51 AML patients with NPM1 mutation A. In 27 patients analysed at diagnosis and after induction treatment, NPM1 mutation A ratios showed a median log(10) reduction of 2.48, which correlated with response to therapy. Among the 51 patients, 21 relapsed and two lost the mutation. We established a sensitive, specific and reproducible assay for routine quantification and monitoring of NPM1 mutation A levels. However, clonal evolution was observed in 9.5% limiting the usefulness of the NPM1 mutation A mutation as a molecular marker in these patients.
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- 2008
29. Rapid and sensitive screening for CEBPA mutations in acute myeloid leukaemia
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Gudrun Mellert, Stefan K. Bohlander, Evelyn Zellmeier, Tobias Benthaus, Purvi M. Kakadia, Michaela Feuring-Buske, Stephanie Schneider, Jan Braess, Karsten Spiekermann, Wolfgang Hiddemann, Annika Dufour, and Friederike Schneider
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Mutation ,medicine.medical_specialty ,Myeloid ,DNA Mutational Analysis ,Cytogenetics ,Hematology ,Gene mutation ,Biology ,medicine.disease_cause ,medicine.disease ,Sensitivity and Specificity ,Leukemia ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,hemic and lymphatic diseases ,Enhancer binding ,CEBPA ,Multiplex polymerase chain reaction ,Cancer research ,medicine ,CCAAT-Enhancer-Binding Protein-alpha ,Humans ,DNA Primers - Abstract
The presence of CCAAT/enhancer binding protein alpha (CEBPA) gene mutations in patients with cytogenetically normal acute myeloid leukaemia (CN-AML) confers a favourable prognosis. Routine screening of all CN-AML patients for CEBPA mutations is therefore important for individual risk-adapted post-remission therapy and requires a fast and easy screening method. CEBPA mutations are distributed over the entire CEBPA gene and the functional and clinical consequences of the different mutations are still largely unknown. Therefore, we developed a multiplex polymerase chain reaction-based fragment length analysis mutation screening method for the entire CEBPA coding region. We initially evaluated our method by analysing 120 CN-AML samples both by fragment analysis and nucleotide sequencing and reached a sensitivity of 100% and a specificity of 90%. 349 CN-AML samples were subsequently screened for CEBPA mutations by fragment length analysis. Among a total of 469 CN-AML patient samples, 58 CEBPA mutations were detected in 38 CN-AML patients (8.1%). In conclusion, we established a fast and sensitive CEBPA mutation screening method suitable for inclusion in routine AML diagnostics.
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- 2008
30. BCR-ABL1-like Acute Lymphoblastic Leukemia Is Associated with IKZF1 and JAK2 Alterations and inferior Outcome in Adults
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Ulrich Mansmann, Natalia Huk, Tobias Herold, Dieter Hoelzer, Luise Hartmann, Martin Neumann, Kathrin Bräundl, Wolfgang Hiddemann, Karsten Spiekermann, Evelyn Zellmeier, Bianka Ksienzyk, Philipp A. Greif, Claudia D. Baldus, Irene Schneider, Nicola Gökbuget, Stephanie Schneider, Nikola P. Konstandin, Vindi Jurinovic, Klaus H. Metzeler, Martin Dreyling, and Stefan K. Bohlander
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medicine.medical_specialty ,education.field_of_study ,biology ,Incidence (epidemiology) ,Immunology ,Population ,Cytogenetics ,Chromosomal translocation ,Cell Biology ,Hematology ,Bioinformatics ,Biochemistry ,Gastroenterology ,Immunophenotyping ,KMT2A ,hemic and lymphatic diseases ,Internal medicine ,medicine ,biology.protein ,Copy-number variation ,Multiplex ligation-dependent probe amplification ,education - Abstract
Background: BCR-ABL1-like (Ph-like) B-precursor acute lymphoblastic leukemia (ALL) displays a gene expression profile closely related to B-precursor ALL with t(9;22)(q34;q11). In addition, Ph-like ALL patients (pts) are characterized by distinct genetic alterations and inferior prognosis in pediatric trials. The purpose of this study was the genetic and clinical characterization of Ph-like ALL in adults. Methods: Affymetrix gene expression profiles (GEP) generated from diagnostic bone marrow samples of 306 adult B-precursor and T-ALL pts (median age 41 years, range 16-84 years) were classified as Ph-like ALL according to published algorithms (Roberts et al., Cancer Cell 2012) and separated from BCR-ABL1-positive and B-other ALL pts (BCR-ABL1-negative; non Ph-like). The incidence and genetic characteristics of the Ph-like subset were analyzed in the overall cohort, whereas clinical and outcome analysis were restricted to B-precursor ALL pts treated within GMALL trials 06/99 and 07/03 (n=107). The median age of this population was 30 (16-64) years. The routine diagnostic work-up included immunophenotyping, fluorescent in situ hybridization (FISH) for BCR-ABL1 and KMT2A (MLL) rearrangements, cytogenetics and molecular analyses of BCR-ABL1 translocations and MLL rearrangements. A subgroup of pts with B-precursor ALL was analyzed for CRLF2 alterations by FISH (n=88) and by RT-PCR for the P2RY8-CRLF2 translocation (n=117). Multiplex ligation-dependent probe amplification (MLPA) for common copy number variations (SALSA MLPA probemix P335-B1) and targeted amplicon sequencing of 131 genes recurrently mutated in ALL were performed in BCR-ABL1 positive (n=30), Ph-like (n=16) and B-other ALL pts (n=23). Results: Of the 306 pts, we classified 26 pts (9%) as Ph-like ALL based on their GEP and the absence of the BCR-ABL1 translocation, corresponding to an incidence of 13% (26/207) in B-precursor ALL and 24% (26/110) among BCR-ABL1-negative B-precursor ALL. Nineteen of 107 B-precursor ALL pts treated within the GMALL trials displayed a Ph-like phenotype. There were no significant differences in baseline characteristics like age, sex, white-cell count, hemoglobin or platelet count and risk group in comparison to the B-other subgroup (n=51). All 19 Ph-like pts showed no MLL rearrangement and 58% belonged to the standard risk group. The complete remission rate after induction was similar for Ph-like and B-other pts (96% vs 100%; p>0.05). At 5 years, the Ph-like ALL subgroup had a lower probability of continuous complete remission (RD: 24% vs 63%; p=0.004) and overall survival (OS: 22% vs 56%; p=0.05) compared to B-other ALL pts. After exclusion of pts with MLL rearrangement from the B-other group (n=11), these differences remained significant (RD: 24% vs 62%; p MLPA and amplicon sequencing revealed specific genetic alterations associated with the Ph-like ALL subgroup (Figure 1). All pts with IGH-CRLF2 (n=6) were identified in the Ph-like subgroup, whereas all pts with P2RY8-CRLF2 (n=2) were found in the B-other group (p0.05, respectively). Additionally, most pts with high CRLF2 expression clustered in the Ph-like ALL subgroup (13/26, 50% vs 8/79, 10% of B-other; p Conclusion: Ph-like ALL in adults is associated with inferior survival in a homogenously treated group of pts. Additionally, molecular analysis revealed distinct genetic alterations identifying this specific ALL subtype. Since gene expression analysis could be difficult to be implemented in routine diagnostics our data suggest, that testing for JAK2 mutations and the IGH-CRLF2 translocation could be options for the diagnosis of the Ph-like subtype. Future treatment strategies should be explored to improve the dismal prognosis for these high risk pts. Figure 1: Distribution of common mutations and deletions in adult B-precursor ALL Figure 1:. Distribution of common mutations and deletions in adult B-precursor ALL Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
31. Quantitative Monitoring By RT-PCR of Molecular Markers on Day +100 after Allogeneic Stem Cell Transplantation Predicts Outcome in Patients with Acute Myeloid Leukemia
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Johanna Tischer, Christina Zeber, Marion Subklewe, Stephanie Schneider, Thomas Koehnke, Nicole Engel, Karsten Spiekermann, Annika Dufour, Dusan Prevalsek, Michael Fiegl, Susanne Fritsch, Markus Pfirrmann, Evelyn Zellmeier, Hans-Jochem Kolb, Roland Reibke, Max Hubmann, and Wolfgang Hiddemann
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Oncology ,medicine.medical_specialty ,business.industry ,Lymphocyte ,Incidence (epidemiology) ,Immunology ,Myeloid leukemia ,Retrospective cohort study ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Minimal residual disease ,Transplantation ,Leukemia ,medicine.anatomical_structure ,hemic and lymphatic diseases ,Internal medicine ,Medicine ,Bone marrow ,business - Abstract
Introduction: Monitoring of minimal residual disease (MRD) is commonly used after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML) patients. Flow cytometry of leukemia-associated aberrant immunophenotypes (LAIP) and chimerism analyses are frequently used tools to monitor MRD. These methods have lower sensitivities and lower relapse detection rates in contrast to molecular markers monitored by quantitative RT-PCR. However, their clinical implications are still limited and only little evidence of single molecular markers is available. We report on a retrospective study of 144 patients with molecular MRD markers transplanted at the Ludwig-Maximilans-University Hospital of Munich-Grosshadern, and analyzed the prognostic values of molecular MRD Monitoring after SCT, baseline risk factors, and follow-up (FU) markers (e.g. chimerism analyses and LAIP). Patients and Methods: Between January 2000 and August 2012, 495 AML patients underwent SCT at our institution. 164 patients had molecular MRD markers and 144 patients had at least one sample with quantitative results prior and/or after SCT, and were included into the analyses. At transplantation, patients had a median age of 48 years (range, 18 -73), and 49 patients were in complete remission, while 95 patients were in a more advanced status of their disease (>CR2). 106 patients received SCT from a HLA matched (10/10) and 38 patients from a HLA mismatched donor. Grafts were obtained either from related (n=60) or unrelated (n=84) donors. Most patients received reduced intensity conditioning (n=142). In 338 bone marrow samples MRD was monitored prior to SCT, on day +30 and on day +100. During the FU period after day +100, MRD was monitored at individual intervals in 429 peripheral blood samples. Quantitative RT-PCR was performed for NPM1 mutation (n=52), MLL-PTD (n=31), RUNX1-RUNX1T1 (n=12), CBFß-MYH11 (n=14), MLL rearrangements (n=20), MDS-EVI1/EVI1 (n=10), and DEK-CAN (n=5). Sensitivities of the different RT-PCRs assays ranged between 10-4 and 10-6. Results: After a median FU of 41 months (range, 4 – 115), 43 patients (30%) relapsed. The MRD levels monitored by RT-PCR prior to SCT and on day +30 after SCT showed no significant impact on relapse-free survival (RFS) and overall survival (OS). At day +100 after SCT, MRD positivity was strongly associated with worse RFS (HR 3.1, p=0.001) and OS (HR 3.2, p=0.004). This was also reflected in a cumulative two-year incidence of relapse of 61% for MRD positive patients versus 15% for MRD negative patients (p Conclusions: Molecular MRD monitoring after SCT might be a useful tool to identify AML patients at high risk for relapse, in contrast to other FU markers, e.g. chimerism analyses and LAIP. In particular, MRD positivity on day +100 after SCT and the switch to MRD positivity during the FU period were significantly associated with worse RFS. As perspective, the individual molecular MRD course might be used as prognostic factor for the guidance of treatment post SCT. Patients with MRD positivity on day +100 after SCT or with a switch to MRD positivity in the FU period may be considered for donor lymphocyte infusions (DLI) or chemotherapeutic interventions, such as hypomethylating agents. Disclosures Subklewe: AMGEN Inc.: Research Funding.
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- 2014
32. Targeted, Deep Sequencing of Adult AML Patients Treated on the AMLCG-2008 Trial Detects Clonal Heterogeneity in 52% of Patients at Initial Diagnosis and Reveals Patterns of Clonal Evolution
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Stephanie Schneider, Nikola P. Konstandin, Philipp A. Greif, Marion Subklewe, Tobias Herold, Karsten Spiekermann, Annika Dufour, Kathrin Bräundl, Stefan K. Bohlander, Michael Fiegl, Bianka Ksienzyk, Klaus H. Metzeler, Jan Braess, Evelyn Zellmeier, Maja Rothenberg-Thurley, and Wolfgang Hiddemann
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Neuroblastoma RAS viral oncogene homolog ,Genetics ,medicine.medical_specialty ,dbSNP ,Immunology ,Cytogenetics ,Cell Biology ,Hematology ,Gene mutation ,Biology ,Biochemistry ,Somatic evolution in cancer ,Deep sequencing ,CEBPA ,medicine ,Allele frequency - Abstract
Background: Recurrent mutations in >100 different genes have been described in AML, but the clinical relevance of most of these alterations has not been defined. Moreover, high-throughput sequencing techniques revealed that AML patients (pts) may harbor multiple, genetically related disease subclones. It is unclear whether clonal heterogeneity at diagnosis also associates with clinical characteristics or outcomes. To address these questions, we set out to characterize a relatively large, uniformly treated patient cohort for mutations in known and putative AML driver genes. Patients and Methods: We studied pretreatment blood or bone marrow specimens from adult AML pts who received high-dose cytarabine-based induction chemotherapy within the German multicenter AMLCG-2008 trial. Sequence variants (single nucleotide variants and insertions/deletions up to approx. 150bp) in 70 genes known to be mutated in AML or other hematologic neoplasms were analyzed by multiplexed amplicon resequencing (Agilent Haloplex; target region, 321 kilobases). Sequencing was performed on an Illumina MiSeq instrument using 2x250bp paired-end reads. A variant allele frequency (VAF) threshold of 2% was set for mutation detection, corresponding to heterozygous mutations present in 4% of cells in a specimen. Variants were classified as known/putative driver mutations, variants of unknown significance, or known germline polymorphisms based on published data (including dbSNP, the Catalogue Of Somatic Mutations In Cancer [COSMIC] and The Cancer Genome Atlas [TCGA]). In patients with more than one single nucleotide variant, the chi square test was used assess if the observed VAFs, adjusted for ploidy, were compatible with the presence of a single clone. Results: Material for genetic analyses was available for 280 of the 396 participants (71%) enrolled on the AMLC-2008 trial. To date, analyses have been completed for 248 pts (130 male, 118 female; median age, 54y; range 19-81y). Updated results for the entire cohort will be presented at the meeting. Mean coverage of target regions was >600-fold, and on average, 98.2% of target bases were covered >30-fold. We detected a total of 914 mutations in 46 genes, including 37 genes mutated in >1 patient (Fig. A). Nine genes (NPM1, FLT3, DNMT3A, NRAS, WT1, IDH2, RUNX1, TET2 and ASXL1) were mutated in >10% of patients (red dashed line in Fig. A). We found a median of 4 mutations per patient (range: 0-10). Of note, only 1 patient had no detectable mutation and no abnormality on cytogenetic analysis. Patients with Intermediate-risk cytogenetics according to the MRC classification harbored a higher number of driver gene mutations (median, 4) compared to patients with MRC Favorable (median, 2 mutations) or Unfavorable (median, 3 mutations) cytogenetics (P When analyzing patterns of co-occurring and mutually exclusive mutations, we confirmed well-known associations (e.g., between CEBPA and GATA2 mutations) and identified novel pairs of mutations that frequently occur in combination and, to our knowledge, have not yet been reported in AML (e.g., ASXL1/STAG2, SRSF2/STAG2). These findings may guide functional studies on the molecular mechanisms of leukemogenesis. We found evidence for clonal heterogeneity in 129 (52%) of 248 pts, based on the presence of mutations with significantly (P Conclusion: Targeted sequencing allowed detection of mutations affecting a panel of known and putative AML driver genes in clinical specimens with high sensitivity. Our data from the AMLCG-2008 patient cohort reveal novel patterns of cooperating gene mutations, and show that the presence of subclonal driver mutations is a frequent event in AML pts. Differentiating between "founding clone" mutations, and subclonal mutations that typically occur later in the disease has implications for choosing targeted therapies aimed at disease eradication. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
33. Genetic Evolution of Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) during Therapy and Relapse: An Exome Sequencing Study of 47 Cases
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Alexander Graf, Evelyn Zellmeier, Luise Hartmann, Wolfgang Hiddemann, Friederike Pastore, Stefan Wiemann, Daniela Schumacher, Bianka Ksienzyk, Stephan Wolf, Helmut Blum, Philipp A. Greif, Claudia D. Baldus, Martin Neumann, Kathrin Bräundl, Stephanie Schneider, Nikola P. Konstandin, Sebastian Vosberg, Stefan K. Bohlander, Karsten Spiekermann, and Klaus H. Metzeler
- Subjects
Oncology ,medicine.medical_specialty ,NPM1 ,Mutation ,Point mutation ,Immunology ,Cytogenetics ,Cancer ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Bioinformatics ,Trisomy 8 ,medicine.disease_cause ,Biochemistry ,Internal medicine ,medicine ,Exome ,Exome sequencing - Abstract
The evolution of acute myeloid leukemia (AML) has been previously described either in studies of large patient cohorts with focus on only a restricted number of AML-associated genes or in smaller series of relapsed patients studied by genome-wide techniques. We set out to comprehensively characterize the genetic evolution in a large AML cohort in order to understand molecular mechanisms of relapse and therapy-resistance. We performed exome-sequencing of matched bone marrow or peripheral blood samples taken at diagnosis, complete remission and relapse from 47 patients with cytogenetically normal AML (CN-AML). Samples were collected within the German Cancer Consortium (DKTK) at the partner sites in Berlin and Munich. The median age at diagnosis was 65y (range: 21-89y). FLT3 internal tandem duplication (ITD) and NPM1 mutation status at diagnosis was available for all but one patient (FLT3-ITD-/NPM1-, n=5; FLT3-ITD+/NPM1-, n=9; FLT3-ITD-/NPM1+, n=16; FLT3-ITD+/NPM1+, n=16). On average, 96% of the target sequence was covered at least 10-fold (minimum coverage defined for variant calling). The following criteria were applied for identification of somatic mutations: Variant allele frequency (VAF) ³20% either at diagnosis or at relapse and VAF Based on cytogenetics and copy number alteration (CNA) analysis of exome data, we detected partial or complete gain/loss of chromosomes. Five patients (11%) acquired chromosomal alterations during disease progression. Trisomy 8 was the only recurrent chromosomal abnormality gained in 3 patients (6%) at relapse. To detect pre-leukemic lesions, we evaluated our exome data for the persistence of mutations in 40 AML-associated driver genes during remission. We limited our analysis to mutations previously reported as confirmed somatic (COSMIC annotation) to avoid confounding with private germline variants. Strikingly, 25/47 (53%) of patients carried non-silent mutations in these genes with VAF>5% (median: 31%, range: 9-75%) at remission (30 mutations in total). In contrast, other mutations (e.g. in FLT3 or NRAS) found in these patients could not be detected at remission, consistent with therapy response. Based on VAF, 23/30 (77%) persistent mutations showed a dynamic pattern over the course of disease with a relative change of >20%, likely due to partial eradication/expansion of leukemic or pre-leukemic clones. Persistent mutations in DNMT3A, TET2, RUNX1 and IDH2 were observed in 28%, 11%, 6% and 4% of patients in our cohort, respectively (Figure 1 B). Among patients with DNMT3A mutation at diagnosis, those with persistent mutations tended to relapse earlier (n=13; median time to relapse 270 days; range: 81-586) than patients without detectable DNMT3A mutations at remission (n=7; median time to relapse 508 days; range: 235-1697; p=0.111). Our findings provide insights into the genetic evolution during the course of disease in a large cohort of relapsed CN-AML. Information about the dynamics of genetic lesions (e.g. persistent or relapse-specific mutations) may have prognostic significance and allow for tailored approaches to treat or to prevent relapse of AML. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.
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- 2014
34. Acute Myeloid Leukemia With Isolated Trisomy 13 Is a Genetically Homogenous Entity With a High Frequency Of Mutations In Genes Encoding Components Of The Splicing Machinery and Extremely Poor Prognosis
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Wolfgang E. Berdel, Thomas Büchner, Annika Dufour, Alexander Graf, Philipp A. Greif, Stefan K. Bohlander, Tobias Herold, Helmut Blum, Karsten Spiekermann, Zlatana Pasalic, Vindi Jurinovic, Cristina Sauerland, Sebastian Vosberg, Evelyn Zellmeier, Bianka Ksienzyk, Purvi M. Kakadia, Bernhard J. Woermann, Stefan Krebs, Klaus H. Metzeler, Stephanie Schneider, Max Hubmann, Ulrich Mansmann, and Wolfgang Hiddemann
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business.industry ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biochemistry ,Gene dosage ,Gene expression profiling ,PTPN11 ,chemistry.chemical_compound ,Exon ,RUNX1 ,chemistry ,hemic and lymphatic diseases ,Cancer research ,Medicine ,business ,Exome sequencing ,Chromosome 13 - Abstract
Acute myeloid leukemia (AML) with isolated trisomy 13 (AML+13) is rare and frequently associated with FAB M0 morphology. The clinical course is not well characterized but according to the ELN classification of intermediate prognosis. Eighty to one-hundred percent of patients (pts) with AML+13 carry mutations in the RUNX1 gene. Over-expression of FLT3 (located on chromosome 13 [chr 13]) due to the additional gene copy on the third chr 13 was proposed as a mechanism of leukemogenesis in AML+13 (gene dosage hypothesis). We set out to characterize the clinical course of AML+13 pts and elucidate their molecular background using whole exome sequencing, targeted resequencing and gene expression profiling. We identified 23 pts with AML+13 enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999) and compared this group to 386 pts without +13 who were classified in the ELN Intermediate-II genetic category. All pts received intensive induction chemotherapy. There was no significant difference in age, white blood cell or platelet count between the two groups. However, LDH levels were significantly (p=.01) lower in the AML+13 group while bone marrow blast percentage was significantly higher (p=.04). Twelve AML+13 pts (52%) reached complete remission, but all relapsed. Relapse-free and overall survival were inferior in the AML+13 group compared to other ELN Intermediate-II pts (median RFS, 9 vs 15 months, p=.01; median OS, 7 vs. 13 months, p=.03). Remission samples from two AML+13 pts were available as normal control for exome sequencing. Using SureSelect human all exon target enrichment (Agilent) followed by 80bp paired-end sequencing on an Illumina GAIIx platform, we were able to identify non-synonymous leukemia-specific mutations affecting, among others, RUNX1, ASXL1, PTPN11 and CEBPZ. Genes identified by exome sequencing and a panel of genes recurringly mutated in AML were studied by targeted amplicon resequencing in all AML+13 pts with available material (16/23; Figure). As described before, a high incidence of RUNX1 mutations (75%) was identified. In addition, we detected mutations in spliceosome components in 14/16 (88%) of AML+13 pts, including SRSF2 codon 95 mutations in 13/16 pts (81%). One patient without SRSF2 mutation showed a mutation in SF3B1. Moreover, recurring mutations were found in ASXL1 (44%) and BCOR (25%), and were associated with RUNX1 and SRSF2 mutations. Interestingly, both pts without mutations in the splicing machinery had mutations in DNMT3A, which were also mutually exclusive with mutations in RUNX1 or ASXL1. Two pts carried mutations in CEBPZ suggesting that CEBPZ is a novel recurringly mutated gene in AML.FigureMutation frequencies in 16 patients with AML+13Figure. Mutation frequencies in 16 patients with AML+13 To further characterize this genetically homogenous subgroup, we compared gene expression profiles of 9 pts with AML+13 with 509 AML pts without +13. We identified 678 (up-regulated 492; down-regulated 186) probe sets as significantly deregulated. Only 59 (8.7%) of these probe sets were localized on chr 13, but of those, 55 were up-regulated and only 4 were down-regulated. Up-regulated probe sets on chr 13 included FOXO1, FLT3 and RB1. The strongest down-regulated probe set on chr 13 belonged to the tumor suppressor gene SPRY2, which is a negative regulator of receptor tyrosine kinases. Gene set enrichment analysis showed significant deregulation of gene sets associated with regulation of transcription and nuclear transport. In summary, our study is the first to show that AML+13 is significantly associated with inferior OS and RFS compared to other intermediate-risk cytogenetic abnormalities in a homogeneously treated cohort. Furthermore, we present evidence that AML+13 leukemias are a genetically quite homogenous subgroup. AML+13 is not only associated with a high rate of RUNX1 mutations but also with mutations in SRFS2, ASXL1 and BCOR. The incidence of mutations in SRSF2 in AML+13 is the highest of any AML subgroup reported so far. In addition, our gene expression data show a homogenous expression profile associated with AML+13. The striking association of a few recurring mutations in AML+13 suggests a biological relationship with synergistic lesions during leukemogenesis. While mutations in RUNX1, ASXL1 and up-regulation of FLT3 were previously reported as markers of poor prognosis in AML, the combination of these lesions might be responsible for the extremely poor outcome of AML+13. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.
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- 2013
35. Dual Inhibition Of PI3K and mTOR Shows Preferential Antileukemic Activity In MLL-Rearranged AML
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Volker Groiß, Sebastian Tiedt, Nadine Sandhöfer, Karsten Spiekermann, Evelyn Zellmeier, Tanja Hinrichsen, Klaus H. Metzeler, Irmela Jeremias, Wolfgang Hiddemann, Oliver Wachter, and Hanns-Georg Klein
- Subjects
Neuroblastoma RAS viral oncogene homolog ,Programmed cell death ,Phosphoinositide 3-kinase ,biology ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Fusion gene ,chemistry.chemical_compound ,chemistry ,hemic and lymphatic diseases ,MK-2206 ,biology.protein ,Cancer research ,Protein kinase B ,STAT5 ,PI3K/AKT/mTOR pathway - Abstract
Background Despite the recent advances and better understanding of the biology of acute myeloid leukemia (AML), only little improvement was achieved in treatment and cure rates. One of the key signaling pathways known to be frequently deregulated in AML is the PI3K/AKT/mTOR signaling axis. In 50-70% of AML patients, its abnormal activation indicated by constitutive phosphorylation of AKT and mTOR, can be observed. Thus, this pathway is seen as a promising target in cancer treatment. In this study we examined the relevance of the PI3K/AKT/mTOR signaling axis in AML by using small molecule inhibitors of AKT, mTOR or both PI3K and mTOR in in vitro and in vivo models. Results With primary AML patient samples we performed propidium iodide (PI) staining and FACS apoptosis analysis. A wide spectrum of cytotoxic activity was observed for single small molecule inhibitors of AKT (MK-2206), mTOR (rapamycin) and a dual PI3K/mTOR inhibitor (BEZ-235) after treatment for 48-72 hours. Dual inhibition of both PI3K and mTOR led to a higher apoptosis induction than single inhibition of AKT or mTOR alone. We could show that patient samples carrying a MLL-translocation, MLL-partial tandem duplication (MLL-PTD) or growth factor signaling (GFS) activating mutations (FLT3, RAS) have a higher sensitivity towards PI3K pathway inhibitors compared to other patient samples (for BEZ-235 median 40.8% vs. 18.8% specific apoptosis). Higher sensitivity in this subgroup was also observed in human-derived AML cell lines. According to their response in apoptosis and proliferation assays we subdivided six AML cell lines into two groups with one group carrying a MLL-AF9 fusion gene plus additional GFS related mutations. The biological response of dual PI3K and mTOR inhibition was well correlated to pathway activity as could be determined by Western Blot and FACS analysis. With 38 MLL-AF9 rearranged patient samples we performed a targeted next generation sequencing approach to further study the relationship between MLL and activating mutations targeting GFS pathways. In 76% of all analyzed patient samples we detected mutations in GFS signaling related genes such as KRAS, NRAS, FLT3 and CBL. KRAS and NRAS mutations were observed much more frequently in MLL-rearranged AML (in 42% and 18% of patients, respectively) than reported in other AML subgroups. Because of the high incidence of RAS mutations we further performed combination treatment studies with a MAPK inhibitor in THP-1 as a model cell line for this patient cohort (carrying a MLL-AF9 fusion and the NRAS G12D mutation). Effects after combination treatment were highly synergistic in apoptosis and proliferation assays (combination index value: 0.05). In vivo activity of PI3K and mTOR inhibiton by BEZ-235 was investigated by using an AML xenograft mouse model derived from MOLM-13 cells, carrying a MLL-AF9 fusion and a FLT3 mutation. To enable in vivo monitoring of the cells after injection, cells were engineered to express GFP and enhanced firefly luciferase (eFFLuc). Treatment led to significantly delayed tumor progression and prolonged overall survival. The tumor load was quantified by in vivo imaging and post mortem FACS analysis and was lower in BEZ-235 treated than in vehicle treated mice. Six hours after administration of the drug, S6rp phosphorylation in leukemic cells was significantly decreased by 50% compared to untreated mice but reached its basal level again 24 hours after administration. Conclusions Our data implicate a possible therapeutic benefit of PI3K/AKT/mTOR inhibition in AML especially in the MLL-rearranged and GFS signaling mutated subgroup. Combination of PI3K/mTOR inhibitors with drugs inhibiting rescue pathways such as the MAPK pathway and Jak2/STAT5 or combination with drugs promoting cell death could improve the therapeutic efficacy of targeted therapies in AML. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
36. A Combined Molecular and Clinical Prognostic Index For Relapse and Survival In Cytogenetically Normal AML (PINA)
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Friederike Pastore, Annika Dufour, Tobias Benthaus, Klaus H. Metzeler, Kati Maharry, Stephanie Schneider, Bianka Ksienzyk, Gudrun Mellert, Evelyn Zellmeier, Purvi M. Kakadia, Michael Unterhalt, Michaela Feuring-Buske, Christian Buske, Jan Braess, Cristina Sauerland, Achim Heinecke, Utz Krug, Wolfgang E. Berdel, Thomas Büchner, Bernhard J. Woermann, Wolfgang Hiddemann, Stefan K. Bohlander, Guido Marcucci, Karsten Spiekermann, Clara D. Bloomfield, and Eva Hoster
- Subjects
Oncology ,medicine.medical_specialty ,Performance status ,business.industry ,Immunology ,Hazard ratio ,Cancer ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Confidence interval ,Group B ,Surgery ,Leukemia ,Internal medicine ,CEBPA ,Cohort ,Medicine ,business - Abstract
Background Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician it is difficult to know how to use the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pre-treatment patient and molecular characteristics. Patients and methods Two prognostic indices for CN-AML, one with regard to overall survival (PINAOS) and the other regarding relapse-free survival (PINARFS) were derived based on a cohort of 669 CN-AML patients treated within the AML Cooperative Group 99 (AMLCG99) study. Results Based on age (median: 60 years [range: 17-85 years]), performance status, white blood count, and presence or absence of NPM1 mutation, biallelic CEBPA mutation, and FLT3-ITD, patients were classified into three risk groups according to PINAOS and PINARFS: 29% of all and 32% of responding patients had low risk (5-year OS 72%; 5-year RFS 55%), 56% and 39% intermediate risk (5-year OS 28%; 5-year RFS 27%), and 15% and 29% high risk disease (5-year OS 3%; 5-year RFS 8%) (Figure 1). PINAOS and PINARFS further subdivided the European LeukemiaNet (ELN) favorable-genetic group as well as the ELN intermediate-I-genetic group. Both, PINAOS and PINARFS were confirmed in a large, independent, and comparable CN-AML cohort of 529 patients from the Cancer and Leukemia Group B (CALGB/Alliance) trials (Figure 2). Conclusions We have developed and validated the first prognostic indices specifically designed for CN-AML patients of all ages combining well-established molecular and clinical variables easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care by risk-adapted therapy of CN-AML. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
37. Analysis of Cooperating Genetic Events in MLLT3-MLL Rearranged Acute Myeloid Leukemia (AML) by Targeted Next-Generation Sequencing of 16 Leukemia-Related Genes Reveals Frequent Mutations Affecting Growth Factor Signalling Pathways and Provides Evidence for Clonal Heterogeneity
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Bianka Ksienzyk, Philipp A. Greif, Purvi M. Kakadia, Marion Subklewe, Hanns-Georg Klein, Karsten Spiekermann, Stefan K. Bohlander, Klaus H. Metzeler, Evelyn Zellmeier, Tanja Hinrichsen, Stephanie Schneider, Nadine Sandhöfer, Annika Dufour, and Wolfgang Hiddemann
- Subjects
Genetics ,Neuroblastoma RAS viral oncogene homolog ,Sanger sequencing ,education.field_of_study ,NPM1 ,Immunology ,Population ,Cell Biology ,Hematology ,Biology ,Gene mutation ,Amplicon ,medicine.disease_cause ,Biochemistry ,symbols.namesake ,Fusion transcript ,hemic and lymphatic diseases ,symbols ,medicine ,KRAS ,education ,neoplasms - Abstract
Abstract 1379 Background: A large number of gene mutations have been recently detected in AML using novel sequencing technologies. We established a rapid, amplicon-based resequencing assay that allows efficient analysis of 16 of the most commonly mutated genes in AML and used it to study a cohort of AML patients (pts) carrying a translocation t(9;11)(p22;q23) (MLLT3-MLL; MLL-AF9). This genetic subgroup, accounting for ∼1% of adult AML, is associated with young age, treatment-related disease, FAB M4/M5 morphology, and an intermediate prognosis. There is limited information on the cooperating genetic lesions in adult AML with t(9;11). Importantly, several widely used murine AML models are based on MLLT3-MLL fusion transcript expression. Thus, a better understanding of the genetic basis of human MLLT3-MLL-rearranged AML is necessary to understand how well these animal models reflect their human counterpart and whether findings from MLLT3-MLL-induced disease are generalizable to other genetic subsets. Patients and Methods: We studied 33 bone marrow samples from adult AML pts with t(9;11)(p22;q23) (age range, 20–71 years; median, 44 years; 21 de novo and 12 therapy-related AML). Mutations in ASXL1, CBL, DNMT3A, FLT3, IDH1, IDH2, KIT, KRAS, NRAS, NPM1, RUNX1, SF3B1, SRSF2, TET2, U2AF1 and WT1 were analyzed from 250ng of genomic DNA using a multiplexed, amplicon-based next-generation sequencing approach (Illumina TruSeq Custom Amplicon assay and MiSeq sequencer). KRAS mutations were independently verified using PCR followed by 454 sequencing (Roche), and NRAS and FLT3 mutations by PCR and melting curve analysis or Sanger sequencing. Results: Per patient, we obtained between 96k and 235k paired-end reads (2×150bp) mapping to the regions of interest, resulting in median coverage depths of the target genes ranging from 180-fold (SRSF2) to >2500-fold (KRAS). Overall, mutations affecting growth factor signalling pathways were detected in 73% of MLLT3-MLL rearranged AML (24/33; Figure): Fourteen pts (42%) carried KRAS mutations mostly affecting the known hotspot codons 12, 13 and 61, 6 pts (18%) had NRAS mutations (mainly at codons 12 or 13), 5 pts had FLT3 mutations (4 tyrosine kinase domain mutations and 1 internal tandem duplication), and 2 pts had mutated CBL. The frequency of RAS gene mutations did not differ significantly between de novo AML and pts with treatment-related disease (P=.26). More than one RAS mutation was found in 7 pts, including pts with 2 (n=3) or 3 (n=1) distinct KRAS mutations, 2 pts with mutations in both NRAS and KRAS, and one patient with 2 NRAS mutations. Interestingly, in some of these pts, one mutation was present in a relatively large proportion of sequencing reads (e.g., patient UPN12 showing a KRAS p.Q61H mutation in 36% of reads, consistent with a heterozygous mutation present in the majority of cells in the specimen), while other coexisting mutations affected a much smaller proportion of reads (in patient UPN12, two different KRAS exon 2 mutations in 5% and 2% of reads, respectively). These results suggest the presence of different subclones within the AML blast population, each carrying a different KRAS mutation. Analyses of follow-up samples are underway to assess changes of clonal architecture over time. Other gene mutations were rarely found in this cytogenetic subgroup of AML: In our 33 pts, we detected 2 ASXL1 mutations, 1 mutation each in TET2, SRSF2 and U2AF1, and no mutations in the other 8 genes we studied. Conclusion: Targeted resequencing using a multiplexed amplicon-based assay is a sensitive and rapid method to screen for mutations in a panel of genes commonly involved in AML pathogenesis. To our knowledge, our report is the first comprehensive analysis of cooperating gene mutations in adult AML with t(9;11)(p22;q23). We demonstrate that MLLT3-MLL-rearranged AML is characterized by frequent mutations in genes involved in growth factor signalling (particularly KRAS and NRAS, mutated in 40% and 18%, respectively, of our MLL-MLLT3 AML cohort compared to only about 5% of unselected AML pts), in the absence of other common AML-related gene mutations. Our results complement recent studies reporting RAS mutations in 45% of infant MLL-rearranged ALL, and functional data from mouse models showing that RAS mutations cooperate with the MLLT3-MLL fusion during leukemogenesis. Finally, our results provide evidence for clonal heterogeneity within MLLT3-MLL rearranged human AML. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2012
38. Quantitative Monitoring of NPM1 Mutation A Minimal Residual Disease Identifies Patients At High Risk for Relapse within the ELN Favorable Risk Group
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Michael Fiegl, Annika Dufour, Evelyn Zellmeier, Thomas Köhnke, Max Hubmann, Karsten Spiekermann, Marion Subklewe, Wolfgang Hiddemann, and Stephanie Schneider
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Oncology ,medicine.medical_specialty ,NPM1 ,business.industry ,medicine.medical_treatment ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Disease ,medicine.disease ,Biochemistry ,Minimal residual disease ,Surgery ,Transplantation ,Leukemia ,hemic and lymphatic diseases ,Internal medicine ,Cohort ,medicine ,business ,Neoadjuvant therapy - Abstract
Abstract 1404 Introduction: Molecular analyses of leukemia-specific markers has led to an improvement of the prognosis evaluation in patients (pts) with acute myeloid leukemia (AML). The European Leukemia Net (ELN) has published a classification which separates different subgroups by cytogenetic and molecular genetic analyses. Nevertheless, there are still pts suffering from disease recurrence within the ELN favorable risk group. To identify these pts at high risk for relapse the monitoring of minimal residual disease (MRD) of leukemia-specific markers could become an important diagnostic tool. In this study the potential of MRD monitoring by quantitative real-time PCR (RT-PCR) of NPM1 A mutation (NPM1 A) at different checkpoints within the ELN favorable risk group of pts with NPM1 A and without FLT3-ITD was investigated. Methods: Pts participating in the AMLCG99, AMLCG2004, and AMLCG2008 trial were prospectively or retrospectively screened for NPM1 mutation and FLT3-ITD by melting curve analyses. 334 pts were screened positive for NPM1 mutation and 262 pts showed a NPM1 A, 78.4 % of all NPM1 mutations. For MRD monitoring a relative RT-PCR was performed in 538 samples of 178 NPM1 A positive pts with a sensitivity of 10-6. MRD was monitored at diagnosis, in aplasia, after induction therapy, after consolidation therapy, and during the follow-up. MRD levels were normalized to the housekeeping gene ABL1 and expressed as a ratio to an internal control of known concentration. Results: In the analysis of the NPM1 A positive and FLT3-ITD negative pts (ELN favorable risk group) 82.5% (n=85) achieved complete remission (CR) after induction therapy. With a median follow-up of 26 (range 1–118) months, 36 (42.9%) pts relapsed within this subgroup. In aplasia, and after induction therapy, pts with a long-lasting remission showed significantly lower NPM1 A ratios in contrast to pts who relapsed during the follow-up. Via Receiver-Operating Curves (ROC) we analyzed the diagnostic power to identify pts at high risk for relapse and determined clinical useable cut-offs at the different checkpoints. ROC were significantly associated with disease recurrence at the checkpoints in aplasia and after induction therapy, but not after consolidation therapy. After induction therapy, a cut-off with a ratio of 0.01 was determined. This cut-off separates the patient cohort into two prognostic groups. NPM1 A MRD levels above the cut-offs result in an increased risk of relapse compared to pts with MRD level below this cut-off. This is reflected in a significantly lower 2-year relapse free survival (RFS) of 18% versus 72% (Figure 1). In 25 pts of this favorable risk group follow-up samples in CR were available for analysis of an upcoming relapse within 100 days of sampling. Only 2 of these pts developed relapse within of the next 100 days, but both pts showed increasing MRD levels prior to relapse. 18 relapse samples were available in this subgroup and interestingly, one patient (5.5%) was NPM1 A negative at relapse. When we further enrolled the FLT3-ITD positive pts into our analyses, not surprisingly we found a negative impact on the RFS of MRD positive and MRD negative pts. Conclusions: Our results confirm the observations of other studies that showed the prognostic impact of NPM1 MRD monitoring by RT-PCR. With the MRD monitoring we could identify pts at high risk for relapse within the ELN favorable risk group. Particularly high MRD levels after the induction therapy were strongly associated with a worse RFS. This and previously published data of others demonstrate that in addition to pre-therapeutic factors, the individual MRD course should be used as prognostic factor for the guidance of treatment and pts with high or increasing levels of MRD should undergo allogeneic stem cell transplantation, if eligible. Disclosures: No relevant conflicts of interest to declare.
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- 2012
39. Disruption of TP53 function by Point Mutations and Deletions Is Associated with An Increased Risk of Disease Progression within Previously Treated, Relapsed Chronic Lymphocytic Leukemia Patients
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Nancy Patten, Boris V. Afanasiev, Tadeusz Robak, Sim Truong, Lin Wu, Galina Salogub, Guillemette Duchateau-Nguyen, Gudrun Mellert, Annika Dufour, Philippe Solal-Celigny, Ming Lin, Giuseppe Palermo, Purvi M. Kakadia, Marco Montillo, Martin Weisser, Ru-Fang Yeh, David Dornan, Anna Dmoszynska, Christian H. Geisler, Stephanie Schneider, Karsten Spiekermann, Stefan K. Bohlander, Evelyn Zellmeier, Wolfgang Hiddemann, John G. Catalano, and Jan Braess
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Biologic marker ,Oncology ,AmpliChip CYP450 Test ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,Point mutation ,Chronic lymphocytic leukemia ,Immunology ,Cell Biology ,Hematology ,Lower risk ,Bioinformatics ,medicine.disease ,Biochemistry ,Frameshift mutation ,Internal medicine ,Medicine ,Missense mutation ,business ,neoplasms ,Fluorescence in situ hybridization - Abstract
Abstract 2445 Chronic lymphocytic leukemia (CLL) patients with a deletion of the TP53 tumor supressor gene located at 17p13 have a poor prognosis in first line chemotherapy regimens. Recent studies indicated somatic TP53 mutations as a prognostic factor in CLL independent of 17p13 deletion status. We aimed to further characterize the prognostic value and the impact of TP53 mutations on progression-free survival (PFS) in the presence and absence of a 17p13 deletion in previously treated and relapsed CLL patients within an international phase III clinical study comparing Fludarabine and Cyclophosphamide with or without Rituximab (FC versus R-FC: REACH trial). We analyzed 457 patients at diagnosis for mutations in the TP53 gene using a combination of a microarray-based resequencing assay (AmpliChip p53 Test, Roche Molecular Systems, USA.) and Sanger sequencing of TP53 exons 2–10. The data were correlated with clinical and biologic markers as well as with interphase fluorescence in situ hybridization (FISH) and with PFS. Association of the clinical data with PFS was assessed by Cox proportional hazard models. To estimate the functional significance of the individual TP53 mutations we used the IARC TP53 database. TP53 mutations (n=60) were detected in 52 of 457 patients (11.4%) and included 42 missense, 4 nonsense, 8 frameshift mutations, 2 in-frame deletions and 4 mutations in splice sites. Among other clinical variables, only 17p13 deletion was associated with TP53 mutations: 27 of 52 TP53 mutated patients had a 17p13 deletion (concordance rate: 52%, Fisher's test p In this large cohort of previously treated CLL patients, complete disruption of TP53 function (by a combination of a 17p13 deletion and a TP53 mutation, through dominant negative TP53 mutations or through multiple TP53 mutations) was associated with a higher risk for disease progression. Prognosis of patients with a single TP53 mutation was not significantly different from patients without TP53 aberrations. It remains to be shown whether CLL patients with a single TP53 mutation are at a higher risk of acquiring additional mutations of TP53 during disease progression. Prognostic stratification of previously treated CLL patients should include a routine molecular TP53 mutational analysis in addition to deletion analysis of the TP53 locus by FISH. Disclosures: Dufour: Roche: Research Funding. Bohlander:Roche: Research Funding. Spiekermann:Roche: Research Funding. Schneider:Roche: Research Funding. Hiddemann:Roche: Research Funding. Truong:Roche: Employment. Patten:Roche: Employment. Wu:Roche: Employment. Dmoszynska:Mundipharma:; Roche: Honoraria. Robak:Centocor Ortho Biotech Research & Development: Research Funding. Geisler:Roche: Speakers Bureau. Dornan:Genentech: Employment. Lin:Genentech: Employment. Yeh:Genentech: Employment. Weisser:Roche: Employment. Duchateau-Nguyen:Roche: Employment. Palermo:Roche: Employment.
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- 2011
40. Low Expression of MiR-34a in Previously Treated Chronic Lymphocytic Leukemia Patients Is Limited to Patients with a Complete Disruption of TP53 Function and Does Not Correlate with MDM2 SNP309
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Galina Salogub, Gudrun Mellert, Martin Weisser, Ru-Fang Yeh, Boris V. Afanasiev, Sim Truong, Guillemette Duchateau-Nguyen, Tadeusz Robak, David Dornan, Ming Lin, Evelyn Zellmeier, Philippe Solal-Celigny, Marco Montillo, Annika Dufour, Nancy Patten, Christian H. Geisler, Karsten Spiekermann, Lin Wu, John G. Catalano, Jan Braess, Wolfgang Hiddemann, Anna Dmoszynska, Purvi M. Kakadia, Stephanie Schneider, Giuseppe Palermo, and Stefan K. Bohlander
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Oncology ,AmpliChip CYP450 Test ,medicine.medical_specialty ,Cyclophosphamide ,Chronic lymphocytic leukemia ,Immunology ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,Biology ,Bioinformatics ,medicine.disease ,Biochemistry ,Chemotherapy regimen ,Fludarabine ,Internal medicine ,Genotype ,medicine ,Rituximab ,medicine.drug - Abstract
Abstract 3521 MicroRNA-34a (miR-34a), a direct downstream target of the tumor suppressor TP53 is upregulated in chronic lymphocytic leukemia (CLL) cells and leads to apoptosis and cell cycle arrest. Previous studies found low miR-34a expression levels in CLL patients with TP53 gene disruptions by either 17p13 deletions or TP53 mutations and this has been linked to chemotherapy resistance and poor prognosis. Alternatively, miR-34a expression levels might be influenced by a single nucleotide polymorphism 309 (SNP 309) in the intronic MDM2 promoter. In this work we retrospectively determined miR-34a expression levels and the TP53 status in previously treated CLL samples enrolled in an international phase III clinical study comparing Fludarabine and Cyclophosphamide with or without Rituximab (FC versus R-FC: REACH trial). MicroRNA profiling data (Affymetrix GeneChip miRNA 1.0 array) and TP53 mutation data (AmpliChip p53 Test and Sanger sequencing) were available for 275 of 546 patients at treatment begin. In this subgroup of patients, genotype data (Illumina 550k HumanHap array) were available for 265 of 275 patients. A Mann-Whitney Wilcoxon test was used to compare distributions across two groups for a continuous variable. Association of the clinical data with progression-free survival (PFS) was assessed by Cox proportional hazard models. Patients were stratified into the following three groups: patients with both a 17p13 deletion and a TP53 mutation and patients with known dominant negative mutations of TP53 (n=21) (complete disruption of TP53), patients with either a 17p13 deletion (n=8) or a TP53 mutation (n=10) (partial disruption of TP53), and patients without TP53 aberrations (n=236) (wildtype TP53 and no 17p13 deletion). The distribution of miR-34a expression levels (log2 transformed) was compared across these groups. Patients with a complete disruption of TP53 function (mean=2.1, sd=2.1) had significantly lower miR-34a expression levels compared to patients without TP53 aberrations (mean=6.8, sd=2.3, p In previously treated CLL patients, only a complete loss of TP53 function correlates with low miR-34a expression levels. MiR-34a expression levels did not demonstrate prognostic significance in CLL patients without TP53 mutations and did not correlate with the presence or absence of MDM2 SNP309. Further studies are warranted to assess the functional and clinical role of miR-34a expression as prognostic factor in patients with a disruption of TP53 function. Disclosures: Dufour: Roche: Research Funding. Bohlander:Roche: Research Funding. Spiekermann:Roche: Research Funding. Schneider:Roche: Research Funding. Hiddemann:Roche: Research Funding. Truong:Roche: Employment. Patten:Roche: Employment. Wu:Roche: Employment. Dmoszynska:Roche: Honoraria. Robak:Centocor Ortho Biotech Research & Development: Research Funding. Geisler:Roche: Speakers Bureau. Dornan:Genentech: Employment. Lin:Genentech: Employment. Yeh:Genentech: Employment. Weisser:Roche: Employment. Duchateau-Nguyen:Roche: Employment. Palermo:Roche: Employment.
- Published
- 2011
41. High Frequency of GATA2 Mutations in Cytogenetically Normal Acute Myeloid Leukemia with Biallelic CEBPA Mutations Identified by Exome Sequencing
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Andreas Hauser, Nikola P. Konstandin, Wolfgang Hiddemann, Alexander Graf, Philipp A. Greif, Wolfgang E. Berdel, Evelyn Zellmeier, Jan Braess, Bernhard J. Woermann, Annika Dufour, Stefan Krebs, Bianka Ksienzyk, Marjan Yaghmaie, Stefan K. Bohlander, Purvi M. Kakadia, Stephanie Schneider, Karsten Spiekermann, Helmut Blum, Tobias Benthaus, and Thomas Büchner
- Subjects
Sanger sequencing ,Genetics ,Mutation ,Genetic heterogeneity ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,Biochemistry ,High Resolution Melt ,symbols.namesake ,CEBPA ,medicine ,symbols ,Missense mutation ,Exome ,Exome sequencing - Abstract
Abstract 72 Cytogenetically normal acute myeloid leukemia (CN-AML) with biallelic CEBPA gene mutations (biCEPBA) represents a distinct genetic entity associated with a favorable clinical outcome (Dufour et al, JCO, 2010; Green et al, JCO, 2010; Pabst et al, Br J Cancer, 2009; Wouters et al, Blood, 2009). Furthermore, biCEBPA mutations are seldomly associated with other known prognostic mutations, like mutated NPM1 or FLT3-ITD. So far, it is not known if other alterations cooperate with the biCEBPA mutations in the process of leukemogenesis. To identify collaborating mutations, we performed whole exome sequencing in five biCEBPA mutated CN-AML patients. We generated at least 5 Gbp of exome sequence for each of the biCEBPA AML samples and for the corresponding remission samples. This allowed us to cover at least 80% of RefSeq coding exon positions with a minimum read depth of 10. Comparison of the AML exome sequence with the remission exome sequence and exclusion of annotated polymorphisms led to the identification of leukemia-specific variants. So far, we were able to confirm between 2 to 10 non-synonymous coding somatic mutations per patient in addition to the previously known biCEBPA mutations using Sanger sequencing. Thus, we detected tumor-specific mutations (nonsense and missense) in a total of 22 genes. Two genes were found recurrently mutated in 2 of the 5 biCEBPA samples: DNMT3A (2/5) and GATA2 (2/5). GATA2 is a zinc finger transcription factor important for haematopoietic stem cell proliferation and normal megakaryocytic development. GATA2 mutations have recently been associated with familial monocytopenia and familial myelodysplastic syndrome (Hsu et al, Blood, 2011; Scott et al, ASH abstract 2010). In the M5 subtype of AML, GATA2 mutations were found at a low frequency of 3.6% (Yan et al, Nature Genetics, 2011). Interestingly, GATA2 is a direct protein interactor and negative regulator of CEBPA. (Huang et al., MCB, 2009; Tong et al, MCB, 2005). Therefore, we determined the frequency of GATA2 mutations in 32 patients with biCEBPA mutant AML by screening all coding exons of GATA2 using high resolution melting curve analysis. Aberrant melting curves were subsequently confirmed by Sanger sequencing. Interestingly, 13 out of 32 (40.6%) biCEBPA patients carried heterozygous missense mutations in GATA2 and strikingly these mutations were all located in the highly conserved N-terminal zinc finger domain of GATA2. The missense mutations A318T and G320D surrounding the C319 which coordiates the zinc atom were recurrently detected in 6 out of 13 biCEBPA patients (3 with A318T and 3 with G320D). Two patients were found to carry each two different mutations in GATA2. 4 out of 13 biCEBPA patients with GATA2 mutations who could be analyzed during molecular remission had lost the GATA2 mutation at remisssion. Furthermore, no GATA2 mutations were found in 38 patients with a monoallelic CEBPA mutation and in 90 CN-AML patients with wildtype CEBPA. We are currently analyzing the functional consequences of these GATA2 mutations. In summary, we describe for the first time the specific association of mutations within the N-terminal zinc finger of GATA2 with biallelic CEBPA mutations in cytogenetically normal AML. Although high throughput sequencing so far has mainly revealed an increasing genetic heterogeneity in AML, our results suggest that there is an association of distinct mutations in defined genetic subgroups of AML. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.
- Published
- 2011
42. A New Molecular and Clinical Prognostic Score for Risk Stratification in CN-AML
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Stefan K. Bohlander, Achim Heinecke, Michael Unterhalt, Michaela Feuring-Buske, Tobias Benthaus, Friederike Schneider, Maria Cristina Sauerland, Christian Buske, Stephanie Schneider, Evelyn Zellmeier, Gudrun Mellert, Thomas Buechner, Annika Dufour, Karsten Spiekermann, Wolfgang E. Berdel, Bernhard Wörmann, Eva Hoster, Jan Braess, and Wolfgang Hiddemann
- Subjects
Oncology ,medicine.medical_specialty ,NPM1 ,Multivariate analysis ,Randomization ,Performance status ,Proportional hazards model ,business.industry ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Surgery ,Internal medicine ,Cohort ,CEBPA ,medicine ,Clinical significance ,business - Abstract
Abstract 2635 Poster Board II-611 Background: Cytogenetically normal acute myeloid leukemia (CN-AML) is associated with an intermediate outcome. A number of clinical and molecular risk factors have been characterized pointing to the heterogeneity of this group. The purpose of the study was to define a prognostic model based on pre-treatment patient characteristics to facilitate choice of therapy by definition of patient groups with different prognoses. Patients and methods: We evaluated four molecular markers (mutations of NPM1, CEBPA, MLL-PTD; FLT3-ITD mutant level; interaction term NPM1 and FLT3-ITD mutant level) and nine clinical parameters (white blood count (WBC), platelet count, hemoglobin level, lactase dehydrogenase (LDH) level, bone marrow blasts, de novo AML vs. non de novo AML, performance status, sex and age) at initial diagnosis in 648 patients with CN-AML treated in the AMLCG (German AML Cooperative Group) 1999 trial. The outcome parameter overall survival (OS) was calculated from randomization to death from any cause or to the latest follow-up date. Event-free survival (EFS) was defined as the period from the start of therapy until lack of a complete remission (CR), relapse of AML after CR or death without relapse. Relapse-free survival (RFS) was determined for responders from the first day of a CR until relapse or death without relapse. Univariate and multivariate Cox regression analyses for OS were performed. All parameters with p'0.05 in multivariate analyses after backward elimination and their regression coefficients were applied in the prognostic score. The minimal p-value approach was used to identify the risk groups with the greatest differences in OS. Results: In our patient cohort 84% had de novo AML. Median age was 60 years (17–85 years) and 70% had an ECOG score ≤1. Median platelet count was 57 G/l (5–643 G/l), median WBC was 18 G/l (0.1–798 G/l) and median hemoglobin level was 9.2 g/dl (4.2–16.4 g/dl). Mutations of NPM1, FLT3-ITD, MLL-PTD and CEBPA were present in 51%, 27%, 8% and 10% of patients, respectively. Median FLT3-ITD mutant level in FLT3-ITD mutated patients was 0.42 (0.02–1.00). Of 648 patients 377 had died. Median OS was 20 months with a median follow-up of 45 months. In the multivariate analyses for OS, the following parameters were significant: age (+10, years, HR: 1.3, p Conclusions: We propose a new prognostic score based on pre-therapeutic clinical and well-established molecular markers that could be easily applied in the routine patient care setting for risk stratification and risk-adapted therapy. Further prospective validation is required to confirm the clinical relevance of this score. Disclosures: Unterhalt: Roche: travel support. Hoster:Roche: travel support.
- Published
- 2009
43. Age-Dependent Frequencies of NPM-1/FLT3-ITD Mutations in Patients with Normal Karyotype AML
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Karsten Spiekermann, Tobias Benthaus, Achim Heinecke, Gudrun Mellert, Eva Hoster, Evelyn Zellmeier, Stefan K. Bohlander, Michael Unterhalt, Friederike Schneider, Stephanie Schneider, Wolfgang Hiddemann, Thomas Buechner, Christian Buske, Michaela Feuring-Buske, Jan Braess, Wolfgang E. Berdel, Annika Dufour, and Maria Cristina Sauerland
- Subjects
medicine.medical_specialty ,NPM1 ,Mutation ,Pediatrics ,business.industry ,Incidence (epidemiology) ,Immunology ,Karyotype ,Cell Biology ,Hematology ,Disease ,Impedance threshold device ,Gene mutation ,medicine.disease_cause ,Biochemistry ,Gastroenterology ,Internal medicine ,medicine ,Chi-square test ,business - Abstract
Background: Long-term survival in NK-AML is influenced by different clinical and molecular markers. Whereas the presence of a NPM-1 mutation is associated with a positive prognostic effect on long-term outcome, the presence of a FLT3-ITD mutation has a negative impact on survival. Interestingly, a significant interaction between NPM-1 and FLT3-ITD mutations has been shown. The positive prognostic impact on clinical outcome was evident predominantly in patients with NK-AML carrying NPM1 gene mutations when FLT3-internal tandem duplications (ITD) were absent. In contrast, the survival in all other groups of NPM-1 and FLT3-ITD combinations was not different so far. A clinical parameter with negative impact on all outcome parameters (OS, EFS, RFS, CR) is patient age at diagnosis. Certainly the worse prognosis in elderly patients is due to adverse patient characteristics and comorbidities. Nevertheless also disease-associated parameters reveal differences between older and younger patients with AML. Therefore we investigated the frequencies of NPM-1/FLT3-ITD mutations in different age groups. Patients and methods: Analyses were based on 803 patients with NK-AML included in the AMLCG (German AML Cooperative Group) 2000 trial until 01/2006. Patient age ranged from 17 to 85 years (median: 60 yrs). Information about the mutation status of NPM-1 and FLT3-ITD mutations at diagnosis was available in 689 patients. Patients were divided into six age groups (1: 17–30yrs; 2: 31–40yrs; 3: 41–50yrs; 4: 51–60yrs; 5: 61–70yrs; 6: 71–85yrs). The incidence of the molecular markers NPM-1 and FLT3-ITD as well as the four NPM-1 and FLT3-ITD combinations were calculated in cross tables (Pearson’s Chi Square test) in the different age groups. Results: In 689 patients with available mutations status we found a significant decrease in the frequency of the two molecular markers with higher age. Whereas the incidence of NPM-1 mutation decreased abruptly in patients >60 yrs [Group 1: 18/28 (64.3%), 2: 35/59 (59.3%), 3: 70/114 (61.4%), 4: 84/143 (58.7%), 5: 98/234 (41.9%), 6: 46/111 (41.4%); p Conclusions: Our data show in a large cohort of 689 patients with NK-AML that the presence of mutations of the molecular markers NPM-1 and FLT3-ITD significantly decreases with age. Consequently the proportion of NPM-1−/FLT3-ITD− patients increases over time. This observation sheds light on the disease biology in older patients with AML. Table 1: Distribution of the NPM-1, FLT3-ITD and the 4 NPM-1/FLT3-ITD subgroups in different age groups age groups NPM-1 + % FLT3-ITD+ (%) NPM-1−/FLT3-ITD−(%) NPM-1+/FLT3-ITD+ (%) NPM-1−/FLT3-ITD+ (%) NPM-1+/FLT3-ITD− (%) 17–30 64.3 50.0 25.0 39.3 10.7 25.0 31–40 59.3 35.6 30.5 25.4 10.2 33.9 41–50 61.4 31.6 28.9 21.9 9.6 39.5 51–60 58.7 32.9 31.5 23.1 9.8 35.7 61–70 41.9 25.6 51.3 18.8 6.8 23.1 71–85 41 4 19.8 50.5 11.7 8.1 29.7 all age groups (%) 50.9 29.0 40.5 20.5 8.5 30.5 p-value < 0.0001*** 0.013* < 0.0001*** 0.020* 0.886 0.024* Figure 1: Proportions of the four NPM-1/FLT3-ITD subgroups in different age groups Figure 1:. Proportions of the four NPM-1/FLT3-ITD subgroups in different age groups
- Published
- 2008
44. Prognostic Impact of Clinical and Molecular Markers in Normal Karyotype AML-Results from the AMLCG 2000 Trial
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Friederike Schneider, Jan Braess, Gudrun Mellert, Christian Buske, Tobias Benthaus, Wolfgang Hiddemann, Thomas Buechner, Michael Unterhalt, Wolfgang E. Berdel, Achim Heinecke, Evelyn Zellmeier, Stephanie Schneider, Karsten Spiekermann, Michaela Feuring-Buske, Stefan K. Bohlander, Eva Hoster, Marietta Rottenkolber, and Annika Dufour
- Subjects
NPM1 ,medicine.medical_specialty ,Univariate analysis ,Mitoxantrone ,Pathology ,Multivariate analysis ,Performance status ,business.industry ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Gastroenterology ,Transplantation ,Regimen ,Internal medicine ,CEBPA ,medicine ,business ,medicine.drug - Abstract
Background: Prognosis of AML is influenced by different clinical and molecular alterations. We performed a multivariate analysis including five molecular markers NPM1, FLT-ITD, CEBPA, FLT-TKD and MLL-PTD combined with clinical parameters at initial diagnosis to refine risk stratification. Patients and methods: Prognostic impact of clinical and molecular parameters in respect to OS, EFS, RFS and CR was assessed in 803 patients with normal karyotype included in the AMLCG (German AML Cooperative Group) 2000 trial until 01/2006. Patients were randomly assigned to treatment with TAD (thioguanine, conventional-dose AraC, daunorubicin) followed by HAM (high-dose AraC, mitoxantrone) or with the double-induction regimen consisting of two courses of HAM (quotation Buechner JCO 2006). Patient age ranged from 17 to 85 years (median: 60 yrs). 51% of patients were male, 49% female. 81% of patients had de novo AML. Performance status was normal or slightly impaired in the majority of patients (71% ECOG 0/1). Median blood counts at diagnosis were: Hb: 9.2 g/dl (4.2–16.4 g/dl); WBC: 16.0 G/l (0.1–798.2 G/l); platelets: 58 G/l (0.02–643 G/l), LDH: 410 U/l (8–14332 U/l) and bone marrow (BM) blasts: 80% (6–100%). Molecular markers’ mutation status and all mentioned clinical parameters were included in univariate analyses. In multivariate analyses only univariate significant parameters were used. Results: In 560 patients with all five molecular markers analyzed by routine molecular techniques at diagnosis the frequency of mutations were the following: 52.7% NPM1+, 29.3% FLT3-ITD+, 6.1% FLT3-TKD+, 7.5% MLL-PTD+ and 7.5% CEBPA+. The majority of analyzed patients (44.1%) showed one single mutation only. About one quarter of patients displayed either none (27.5%) or two (26.2%) mutations. A minority of 2.1% had 3 mutations, whereas the combination of four or all five molecular alterations was not found. The most frequent single mutation was NPM1 (28.4%), followed by FLT3-ITD (5.4%), CEBPA (4.8%), MLL-PTD (4.6%) and FLT3-TKD (0.9%). The combination of FLT3-ITD and NPM1 was detected in 18.8% of patients. Complete remission (CR) rate was 65.1%. Median overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) were 19.3, 7.7 and 17.2 months. Multivariate analyses identified the following parameters to have significant impact on prognosis. OS: NPM1, FLT3-ITD, WBC, age (p Conclusions: Our data show in a large cohort of 560 patients that at least one molecular marker can be identified in 72.5% of patients with NK-AML. The NPM1 mutation and age are the only parameters with an independent impact on all outcome parameters (OS, EFS, RFS, CR). These data provide the basis for a prognostic model in NK-AML that can be used for risk stratification and selection of patients that will benefit from allogeneic stem cell transplantation.
- Published
- 2007
45. A Rapid and Sensitive Method for Large-Scale Screening of CEBPA Mutations in AML Patients with Normal Karyotype
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Tobias Benthaus, Wolfgang Hiddemann, Annika Dufour, Gudrun Mellert, Karsten Spiekermann, and Evelyn Zellmeier
- Subjects
Genetics ,Immunology ,Nonsense mutation ,Cell Biology ,Hematology ,Amplicon ,Biology ,Biochemistry ,Molecular biology ,Gene duplication ,CEBPA ,Multiplex polymerase chain reaction ,Coding region ,Multiplex ,Primer (molecular biology) - Abstract
In 45% of patients with de novo acute myeloid leukemia (AML) no cytogenetic abnormalities can be detected (normal karyotype AML, NK-AML). Recently, several molecular markers with prognostic significance have been described which define distinct subgroups of NK-AML patients. Mutations in the CEBPA gene have shown to occur at about 8% of NK-AML in Western countries and confer a favorable prognosis. The C/EBPα protein, a member of the family of basic region leucine zipper (bZIP) transcriptional regulators, is important for normal granulocytic differentiation and is frequently disrupted in AML. We retrospectively analyzed bone-marrow samples of 442 patients with de novo NK-AML for the presence of CEBPA mutations and established a fast and sensitive screening method. A multiplex-PCR-gene scanning assay for combined detection of CEBPA and NPM mutation has recently been described in which, however, the primers did not cover the whole CEBPA gene. CEBPA mutations have been found to be distributed over the entire CEBPA gene and their functional and clinical consequences are not yet clear. Therefore, we designed a rapid CEBPA specific multiplex PCR-gene scanning assay covering the entire coding region of the CEBPA gene. Four primer pairs were designed, fluorescently labeled and included in 2 multiplex PCR reactions. The PCR products were electrophoresed on a genetic analyzer and the amplicon sizes were compared to wildtype CEBPA of U937 cell line by fragment length analysis. In order to evaluate our method, we analyzed 120 patient samples in parallel by both multiplex PCR and sequencing analysis. Using sequencing analysis as a gold standard, all of the CEBPA mutations could be detected by multiplex PCR and fragment length analysis. Thus, our multiplex PCR assay reached a sensitivity of 100%. The specificity was 89% due to the false positive detection of a 6 basepair duplication polymorphism in 2.5% of patients (Wouters BJ et al, Blood, 2007). 322 patient samples were subsequently screened for CEBPA mutations by the multiplex PCR assay. In case of alterations in the fragment length analysis, the relevant CEBPA region was sequenced to identify the exact type of mutation. Among 442 patients with NK-AML, 32 patients (7%) showed CEBPA mutations. Taken together, we identified 47 mutations in 32 patients, of which 17 patients had a single CEBPA mutation and 15 patients had more than one CEBPA mutation. We identified 30 out of frame insertion/deletion nonsense mutations resulting in an N-terminal stopcodon and 14 in frame insertion/deletion mutations as well as 3 out of frame insertion/deletion mutations in the C-terminal bZIP region. The only limitation of this method might be that single basepair substitutions, which do not affect the length of the amplicon, cannot be detected. Substitutions in the CEBPA gene are, however, rare events and often silent. In 120 sequenced AML patients we did not find any non-silent substitution. We established a fast and sensitive screening method suitable for large-scale detection of CEBPA mutation and applicable for inclusion in routine AML diagnostics. This is so far the largest reported analysis of CEBPA mutations in patients with NK-AML and might provide further insights into the functional and clinical relevance of the different types of CEBPA mutations and their correlation to other molecular markers in NK-AML.
- Published
- 2007
46. FLT3-ITD but Not FLT3-TKD Mutations Have Major Prognostic Impact in Normal Karyotype AML
- Author
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Eva Hoster, Michaela Feuring-Buske, Annika Dufour, Stefan K. Bohlander, Evelyn Zellmeier, Wolfgang E. Berdel, Susanne Fritsch, Maria Cristina Sauerland, Karsten Spiekermann, Michael Unterhalt, Gudrun Mellert, Achim Heinecke, Christian Buske, Bernhard Wörmann, Friederike Schneider, Wolfgang Hiddemann, Jan Braess, Thomas Buechner, and Stephanie Schneider
- Subjects
Oncology ,NPM1 ,medicine.medical_specialty ,Mutation ,Point mutation ,Immunology ,hemic and immune systems ,Karyotype ,Cell Biology ,Hematology ,Biology ,Bioinformatics ,medicine.disease_cause ,Biochemistry ,fluids and secretions ,hemic and lymphatic diseases ,Internal medicine ,embryonic structures ,Cohort ,medicine ,In patient ,Gene ,Flt3 itd - Abstract
Background: Approximately 45% of AML patients have a normal karyptype (NK-AML) and an intermediate clinical prognosis. As only 20–42% of these patients show long-term survival, it is important to identify prognostic markers to distinguish patients’ outcome more precisely. Mutations in the FLT3 gene such as internal tandem duplications (ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain (TKD) are the second most common abnormalties in AML patients. For FLT3-ITD it is well known that patients have an unfavourable prognosis. Up to now there are not enough reliable data to determine the prognostic impact of FLT3-TKD mutations. Patients and Methods: We have investigated the prevalence of FLT3-TKD mutations in a cohort of 803 cytogenetically normal AML (NK-AML) patients and its possible prognostic significance. At diagnosis the mutation status of FLT3 (ITD and TKD) and the NPM1 gene were analyzed by routine molecular techniques. Results: The median age of all patients was 60 years and the median observation time of survivors 23.2 months. Results of the mutation status’ of FLT3-ITD, FLT3-TKD and NPM1 were available in 757/803 (94.3%), 683/803 (85.1%) and 696/803 (86.7%) patients, respectively. FLT3-ITD, FLT3-TKD and NPM1 mutations were found in 222 (29.3%), 46 (6.7%) and 354 (50.9%) of all analyzed patients, respectively. We could not detect any influence of the FLT3-TKD mutation on OS (p= 0.753), RFS (p= 0.229), EFS (p= 0.835), CR (p= 0.168) and on d16 blast count (p= 0.696). In most patients FLT3-ITD and TKD mutations were mutually exclusive, although a minority of 8/674 patients (1.2%) carried both mutations. FLT3-TKD mutations were more frequently found in patients with NPM1 mutations compared to NPM1-negative patients (9.04% vs. 3.74%; p= 0.008). In contrast to FLT3-ITD mutations FLT3-TKD mutation had no prognostic impact in NPM1 positive AML cases. Conclusions: In our study in a large cohort of 803 NK-AML patients we could not detect any prognostic impact of FLT3-TKD mutations. Although FLT3-ITD and TKD mutations have both transforming potential in vitro and in vivo mouse models, the clinical impact of both mutations shows striking differences. Further studies with FLT3-PTK inhibitors will clarify the pathogenetic relevance of these mutations in AML.
- Published
- 2007
47. Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia
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Susanne Amler, Michael Fiegl, Stefan K. Bohlander, Wolfgang E. Berdel, Tobias Herold, Stephanie Schneider, Thomas Büchner, Marion Subklewe, Andreas Faldum, Maria Cristina Sauerland, Evelyn Zellmeier, Philipp A. Greif, Karsten Spiekermann, Klaus H. Metzeler, Annika Dufour, Luise Hartmann, Kathrin Bräundl, Maja Rothenberg-Thurley, Bernhard Wörmann, Jan Braess, Wolfgang Hiddemann, Utz Krug, Bianka Ksienzyk, Dennis Görlich, and Nikola P. Konstandin
- Subjects
Adult ,Male ,0301 basic medicine ,NPM1 ,Myeloid ,Adolescent ,DNA Mutational Analysis ,Immunology ,Biology ,Gene mutation ,Bioinformatics ,Biochemistry ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Gene Frequency ,Risk Factors ,CEBPA ,medicine ,Humans ,Genetic Predisposition to Disease ,Survival analysis ,Aged ,Aged, 80 and over ,Age Factors ,Myeloid leukemia ,Adult Acute Myeloid Leukemia ,Cell Biology ,Hematology ,Middle Aged ,Prognosis ,medicine.disease ,Survival Analysis ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cytogenetic Analysis ,Multivariate Analysis ,Mutation ,Female ,Nucleophosmin - Abstract
The clinical and prognostic relevance of many recently identified driver gene mutations in adult acute myeloid leukemia (AML) is poorly defined. We sequenced the coding regions or hotspot areas of 68 recurrently mutated genes in a cohort of 664 patients aged 18 to 86 years treated on 2 phase 3 trials of the German AML Cooperative Group (AMLCG). The median number of 4 mutations per patient varied according to cytogenetic subgroup, age, and history of previous hematologic disorder or antineoplastic therapy. We found patterns of significantly comutated driver genes suggesting functional synergism. Conversely, we identified 8 virtually nonoverlapping patient subgroups, jointly comprising 78% of AML patients, that are defined by mutually exclusive genetic alterations. These subgroups, likely representing distinct underlying pathways of leukemogenesis, show widely divergent outcomes. Furthermore, we provide detailed information on associations between gene mutations, clinical patient characteristics, and therapeutic outcomes in this large cohort of uniformly treated AML patients. In multivariate analyses including a comprehensive set of molecular and clinical variables, we identified DNMT3A and RUNX1 mutations as important predictors of shorter overall survival (OS) in AML patients
48. Combined molecular and clinical prognostic index for relapse and survival in cytogenetically normal acute myeloid leukemia.
- Author
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Pastore F, Dufour A, Benthaus T, Metzeler KH, Maharry KS, Schneider S, Ksienzyk B, Mellert G, Zellmeier E, Kakadia PM, Unterhalt M, Feuring-Buske M, Buske C, Braess J, Sauerland MC, Heinecke A, Krug U, Berdel WE, Buechner T, Woermann B, Hiddemann W, Bohlander SK, Marcucci G, Spiekermann K, Bloomfield CD, and Hoster E
- Subjects
- Adolescent, Adult, Age Factors, Aged, Aged, 80 and over, CCAAT-Enhancer-Binding Proteins genetics, DNA Mutational Analysis, Disease-Free Survival, Female, Genetic Predisposition to Disease, Germany, Humans, Kaplan-Meier Estimate, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute mortality, Leukemia, Myeloid, Acute pathology, Male, Middle Aged, Mutation, Nuclear Proteins genetics, Nucleophosmin, Phenotype, Predictive Value of Tests, Proportional Hazards Models, Recurrence, Reproducibility of Results, Risk Assessment, Risk Factors, Time Factors, Treatment Outcome, Young Adult, fms-Like Tyrosine Kinase 3 genetics, Cytogenetic Analysis, Decision Support Techniques, Leukemia, Myeloid, Acute diagnosis, Leukemia, Myeloid, Acute drug therapy
- Abstract
Purpose: Cytogenetically normal (CN) acute myeloid leukemia (AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician, it is difficult to summarize the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pretreatment patient and disease characteristics., Patients and Methods: Two prognostic indices for CN-AML (PINA), one regarding overall survival (OS; PINAOS) and the other regarding relapse-free survival (RFS; PINARFS), were derived from data of 572 patients with CN-AML treated within the AML Cooperative Group 99 study (www.aml-score.org)., Results: On the basis of age (median, 60 years; range, 17 to 85 years), performance status, WBC count, and mutation status of NPM1, CEBPA, and FLT3-internal tandem duplication, patients were classified into the following three risk groups according to PINAOS and PINARFS: 29% of all patients and 32% of 381 responding patients had low-risk disease (5-year OS, 74%; 5-year RFS, 55%); 56% of all patients and 39% of responding patients had intermediate-risk disease (5-year OS, 28%; 5-year RFS, 27%), and 15% of all patients and 29% of responding patients had high-risk disease (5-year OS, 3%; 5-year RFS, 5%), respectively. PINAOS and PINARFS stratified outcome within European LeukemiaNet genetic groups. Both indices were confirmed on independent data from Cancer and Leukemia Group B/Alliance trials., Conclusion: We have developed and validated, to our knowledge, the first prognostic indices specifically designed for adult patients of all ages with CN-AML that combine well-established molecular and clinical variables and that are easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care through risk-adapted therapy of CN-AML., (© 2014 by American Society of Clinical Oncology.)
- Published
- 2014
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