Publications by authors named "Maja Rothenberg-Thurley"

16 Publications

  • Page 1 of 1

Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia.

Leukemia 2020 10 1;34(10):2621-2634. Epub 2020 May 1.

Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukemia remain largely unexplored. In this study, we characterized mutations in SRSF2, U2AF1, and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile which is highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline, we accurately detected aberrant splicing in splicing factor mutated samples. A large proportion of differentially used junctions were novel, including several junctions in leukemia-associated genes. In SRSF2(P95H) mutants, we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Furthermore, we observed a validated impact on overall survival for two junctions overused in SRSF2(P95H) mutants. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.
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http://dx.doi.org/10.1038/s41375-020-0839-4DOI Listing
October 2020

Validation and refinement of the revised 2017 European LeukemiaNet genetic risk stratification of acute myeloid leukemia.

Leukemia 2020 12 30;34(12):3161-3172. Epub 2020 Mar 30.

Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

The revised 2017 European LeukemiaNet (ELN) recommendations for genetic risk stratification of acute myeloid leukemia have been widely adopted, but have not yet been validated in large cohorts of AML patients. We studied 1116 newly diagnosed AML patients (age range, 18-86 years) who had received induction chemotherapy. Among 771 patients not selected by genetics, the ELN-2017 classification re-assigned 26.5% of patients into a more favorable or, more commonly, a more adverse-risk group compared with the ELN-2010 recommendations. Forty percent of the cohort, and 51% of patients ≥60 years, were classified as adverse-risk by ELN-2017. In 599 patients <60 years, estimated 5-year overall survival (OS) was 64% for ELN-2017 favorable, 42% for intermediate-risk and 20% for adverse-risk patients. Among 517 patients aged ≥60 years, corresponding 5-year OS rates were 37, 16, and 6%. Patients with biallelic CEBPA mutations or inv(16) had particularly favorable outcomes, while patients with mutated TP53 and a complex karyotype had especially poor prognosis. DNMT3A mutations associated with inferior OS within each ELN-2017 risk group. Our results validate the prognostic significance of the revised ELN-2017 risk classification in AML patients receiving induction chemotherapy across a broad age range. Further refinement of the ELN-2017 risk classification is possible.
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http://dx.doi.org/10.1038/s41375-020-0806-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685975PMC
December 2020

Allelic Imbalance of Recurrently Mutated Genes in Acute Myeloid Leukaemia.

Sci Rep 2019 08 13;9(1):11796. Epub 2019 Aug 13.

Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

The patho-mechanism of somatic driver mutations in cancer usually involves transcription, but the proportion of mutations and wild-type alleles transcribed from DNA to RNA is largely unknown. We systematically compared the variant allele frequencies of recurrently mutated genes in DNA and RNA sequencing data of 246 acute myeloid leukaemia (AML) patients. We observed that 95% of all detected variants were transcribed while the rest were not detectable in RNA sequencing with a minimum read-depth cut-off (10x). Our analysis focusing on 11 genes harbouring recurring mutations demonstrated allelic imbalance (AI) in most patients. GATA2, RUNX1, TET2, SRSF2, IDH2, PTPN11, WT1, NPM1 and CEBPA showed significant AIs. While the effect size was small in general, GATA2 exhibited the largest allelic imbalance. By pooling heterogeneous data from three independent AML cohorts with paired DNA and RNA sequencing (N = 253), we could validate the preferential transcription of GATA2-mutated alleles. Differential expression analysis of the genes with significant AI showed no significant differential gene and isoform expression for the mutated genes, between mutated and wild-type patients. In conclusion, our analyses identified AI in nine out of eleven recurrently mutated genes. AI might be a common phenomenon in AML which potentially contributes to leukaemogenesis.
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http://dx.doi.org/10.1038/s41598-019-48167-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692371PMC
August 2019

Loss of KDM6A confers drug resistance in acute myeloid leukemia.

Leukemia 2020 01 14;34(1):50-62. Epub 2019 Jun 14.

Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.
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http://dx.doi.org/10.1038/s41375-019-0497-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214274PMC
January 2020

The neuropeptide receptor calcitonin receptor-like (CALCRL) is a potential therapeutic target in acute myeloid leukemia.

Leukemia 2019 12 10;33(12):2830-2841. Epub 2019 Jun 10.

Department of Medicine A, University Hospital Münster, Münster, Germany.

Calcitonin receptor-like (CALCRL) is a G-protein-coupled neuropeptide receptor involved in the regulation of blood pressure, angiogenesis, cell proliferation, and apoptosis, and is currently emerging as a novel target for the treatment of migraine. This study characterizes the role of CALCRL in acute myeloid leukemia (AML). We analyzed CALCRL expression in collectively more than 1500 well-characterized AML patients from five international cohorts (AMLCG, HOVON, TCGA, Leucegene, and UKM) and evaluated associations with survival. In the AMLCG analytic cohort, increasing transcript levels of CALCRL were associated with decreasing complete remission rates (71.5%, 53.7%, 49.6% for low, intermediate, high CALCRL expression), 5-year overall (43.1%, 26.2%, 7.1%), and event-free survival (29.9%, 15.8%, 4.7%) (all P < 0.001). CALCRL levels remained associated with all endpoints on multivariable regression analyses. The prognostic impact was confirmed in all validation sets. Genes highly expressed in CALCRL AML were significantly enriched in leukemic stem cell signatures and CALCRL levels were positively linked to the engraftment capacity of primary patient samples in immunocompromised mice. CRISPR-Cas9-mediated knockout of CALCRL significantly impaired colony formation in human myeloid leukemia cell lines. Overall, our study demonstrates that CALCRL predicts outcome beyond existing risk factors and is a potential therapeutic target in AML.
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http://dx.doi.org/10.1038/s41375-019-0505-xDOI Listing
December 2019

Genetic heterogeneity of cytogenetically normal AML with mutations of .

Blood Adv 2018 10;2(20):2724-2731

Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, Ludwig-Maximilians-Universität, Munich, Germany.

Biallelic mutations of the CCAAT/enhancer binding protein α () gene define a distinct genetic entity of acute myeloid leukemia (AML) with favorable prognosis. The presence of and mutations that are specifically associated with this subgroup but not mutated in all samples suggests a genetic heterogeneity of bi-mutated AML. We characterized the mutational landscape of -mutated cytogenetically normal AML by targeted amplicon resequencing. We analyzed 48 biallelically mutated (bi), 32 monoallelically mutated (mo), and 287 wild-type (wt) patient samples from German AML Cooperative Group studies or registry. Targeted sequencing of 42 genes revealed that mo patients had significantly more additional mutations and additional mutated genes than bi patients. Within the group of bi patients, we identified 2 genetic subgroups defined by the presence or absence of mutations in chromatin/DNA modifiers (C), cohesin complex (C), and splicing (S) genes: bi (25/48 [52%]) and bi (23/48 [48%]). Equivalent subgroups were identified in 51 bi patients from the Cancer Genome Project. Patients in the bi group were significantly older and had poorer overall survival and lower complete remission rates following intensive chemotherapy regimens compared with patients in the bi group. Patients with available remission samples from the bi group cleared the bi mutations, but most had persisting CCS mutations in complete remission, suggesting the presence of a preleukemic clone. In conclusion, CCS mutations define a distinct biological subgroup of bi AML that might refine prognostic classification of AML. This trial was registered at www.clinicaltrials.gov as #NCT00266136 and NCT01382147.
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http://dx.doi.org/10.1182/bloodadvances.2018016840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199648PMC
October 2018

Mediation analysis reveals common mechanisms of RUNX1 point mutations and RUNX1/RUNX1T1 fusions influencing survival of patients with acute myeloid leukemia.

Sci Rep 2018 07 26;8(1):11293. Epub 2018 Jul 26.

Institute for Medical Information Processing, Biometry and Epidemiology, LMU Munich, Munich, Germany.

Alterations of RUNX1 in acute myeloid leukemia (AML) are associated with either a more favorable outcome in the case of the RUNX1/RUNX1T1 fusion or unfavorable prognosis in the case of point mutations. In this project we aimed to identify genes responsible for the observed differences in outcome that are common to both RUNX1 alterations. Analyzing four AML gene expression data sets (n = 1514), a total of 80 patients with RUNX1/RUNX1T1 and 156 patients with point mutations in RUNX1 were compared. Using the statistical tool of mediation analysis we identified the genes CD109, HOPX, and KIAA0125 as candidates for mediator genes. In an analysis of an independent validation cohort, KIAA0125 again showed a significant influence with respect to the impact of the RUNX1/RUNX1T1 fusion. While there were no significant results for the other two genes in this smaller validation cohort, the observed relations linked with mediation effects (i.e., those between alterations, gene expression and survival) were almost without exception as strong as in the main analysis. Our analysis demonstrates that mediation analysis is a powerful tool in the identification of regulative networks in AML subgroups and could be further used to characterize the influence of genetic alterations.
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http://dx.doi.org/10.1038/s41598-018-29593-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062501PMC
July 2018

Clonal heterogeneity of -ITD detected by high-throughput amplicon sequencing correlates with adverse prognosis in acute myeloid leukemia.

Oncotarget 2018 Jul 10;9(53):30128-30145. Epub 2018 Jul 10.

Experimental Leukemia and Lymphoma Research, Department of Medicine III, University Hospital, LMU Munich, Munich, Germany.

In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of are frequent mutations associated with unfavorable prognosis. At diagnosis, the ITD status is routinely assessed by fragment analysis, providing information about the length but not the position and sequence of the ITD. To overcome this limitation, we performed cDNA-based high-throughput amplicon sequencing (HTAS) in 250 ITD positive AML patients, treated on German AML Cooperative Group (AMLCG) trials. ITD status determined by routine diagnostics was confirmed by HTAS in 242 out of 250 patients (97%). The total number of ITDs detected by HTAS was higher than in routine diagnostics ( = 312 vs. = 274). In particular, HTAS detected a higher number of ITDs per patient compared to fragment analysis, indicating higher sensitivity for subclonal ITDs. Patients with more than one ITD according to HTAS had a significantly shorter overall and relapse free survival. There was a close correlation between ITD mRNA levels in fragment analysis and variant allele frequency in HTAS. However, the abundance of long ITDs (≥75nt) was underestimated by HTAS, as the size of the ITD affected the mappability of the corresponding sequence reads. In summary, this study demonstrates that HTAS is a feasible approach for ITD detection in AML patients, delivering length, position, sequence and mutational burden of this alteration in a single assay with high sensitivity. Our findings provide insights into the clonal architecture of ITD positive AML and have clinical implications.
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http://dx.doi.org/10.18632/oncotarget.25729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059024PMC
July 2018

Gene mutations and clonal architecture in myelodysplastic syndromes and changes upon progression to acute myeloid leukaemia and under treatment.

Br J Haematol 2018 09 5;182(6):830-842. Epub 2018 Jul 5.

Department of Medicine I, Medical Centre - University of Freiburg, Freiburg, Germany.

Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.
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http://dx.doi.org/10.1111/bjh.15461DOI Listing
September 2018

Genetics of acute myeloid leukemia in the elderly: mutation spectrum and clinical impact in intensively treated patients aged 75 years or older.

Haematologica 2018 11 14;103(11):1853-1861. Epub 2018 Jun 14.

Laboratory for Leukemia Diagnostics, Department of Medicine III, University Hospital, LMU Munich, Germany

A cute myeloid leukemia is a disease of the elderly (median age at diagnosis, 65-70 years). The prognosis of older acute myeloid leukemia patients is generally poor. While genetic markers have become important tools for risk stratification and treatment selection in young and middle-aged patients, their applicability in very old patients is less clear. We sought to validate existing genetic risk classification systems and identify additional factors associated with outcomes in intensively treated patients aged ≥75 years. In 151 patients who received induction chemotherapy in the AMLCG-1999 trial, we investigated recurrently mutated genes using a targeted sequencing assay covering 64 genes. The median number of mutated genes per patient was four. The most commonly mutated genes were (42%), (35%), (32%), (25%) and (21%). The complete remission rate was 44% and the 3-year survival was 21% for the entire cohort. While adverse-risk cytogenetics (MRC classification) were associated with shorter overall survival (=0.001), and -ITD mutations (present in 18%) did not have a significant impact on overall survival. Notably, none of the 13 -mutated patients (9%) reached complete remission. Consequently, the overall survival of this subgroup was significantly shorter than that of -wildtype patients (<0.001). In summary, even among very old, intensively treated, acute myeloid leukemia patients, adverse-risk cytogenetics predict inferior survival. The spectrum and relevance of driver gene mutations in elderly patients differs from that in younger patients. Our data implicate mutations as a novel marker for chemorefractory disease and inferior prognosis. (AMLCG-1999 trial: clinicaltrials.gov identifier, NCT00266136).
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http://dx.doi.org/10.3324/haematol.2018.191536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278991PMC
November 2018

Persistence of pre-leukemic clones during first remission and risk of relapse in acute myeloid leukemia.

Leukemia 2018 07 23;32(7):1598-1608. Epub 2018 Feb 23.

Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, LMU Munich, Munich, Germany.

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 < 0.0001) and, in multivariate analyses adjusting for age, genetic risk, and allogeneic transplantation, with inferior relapse-free survival (hazard ratio (HR), 2.34; p = 0.0039) and overall survival (HR, 2.14; p = 0.036). Patients with persisting mutations had a higher cumulative incidence of relapse before, but not after allogeneic stem cell transplantation. Our work underlines the relevance of mutation persistence during first remission as a novel risk factor in AML. Persistence of pre-leukemic clones may contribute to the inferior outcome of elderly AML patients. Allogeneic transplantation abrogated the increased relapse risk associated with persisting pre-leukemic clones, suggesting that mutation persistence may guide post-remission treatment.
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http://dx.doi.org/10.1038/s41375-018-0034-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035153PMC
July 2018

A 29-gene and cytogenetic score for the prediction of resistance to induction treatment in acute myeloid leukemia.

Haematologica 2018 03 14;103(3):456-465. Epub 2017 Dec 14.

Department of Internal Medicine III, University of Munich, Germany.

Primary therapy resistance is a major problem in acute myeloid leukemia treatment. We set out to develop a powerful and robust predictor for therapy resistance for intensively treated adult patients. We used two large gene expression data sets (n=856) to develop a predictor of therapy resistance, which was validated in an independent cohort analyzed by RNA sequencing (n=250). In addition to gene expression markers, standard clinical and laboratory variables as well as the mutation status of 68 genes were considered during construction of the model. The final predictor (PS29MRC) consisted of 29 gene expression markers and a cytogenetic risk classification. A continuous predictor is calculated as a weighted linear sum of the individual variables. In addition, a cut off was defined to divide patients into a high-risk and a low-risk group for resistant disease. PS29MRC was highly significant in the validation set, both as a continuous score (OR=2.39, =8.63·10, AUC=0.76) and as a dichotomous classifier (OR=8.03, =4.29·10); accuracy was 77%. In multivariable models, only mutation, age and PS29MRC (continuous: OR=1.75, =0.0011; dichotomous: OR=4.44, =0.00021) were left as significant variables. PS29MRC dominated all models when compared with currently used predictors, and also predicted overall survival independently of established markers. When integrated into the European LeukemiaNet (ELN) 2017 genetic risk stratification, four groups (median survival of 8, 18, 41 months, and not reached) could be defined (=4.01·10). PS29MRC will make it possible to design trials which stratify induction treatment according to the probability of response, and refines the ELN 2017 classification.
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http://dx.doi.org/10.3324/haematol.2017.178442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830382PMC
March 2018

Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia.

Blood 2016 08 10;128(5):686-98. Epub 2016 Jun 10.

Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, Ludwig-Maximilans-Universität, Munich, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany;

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 <60 years, and particularly in those with intermediate-risk cytogenetics. NPM1 mutations in the absence of FLT3-ITD, mutated TP53, and biallelic CEBPA mutations were identified as important molecular prognosticators of OS irrespective of patient age. In summary, our study provides a comprehensive overview of the spectrum, clinical associations, and prognostic relevance of recurrent driver gene mutations in a large cohort representing a broad spectrum and age range of intensively treated AML patients.
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http://dx.doi.org/10.1182/blood-2016-01-693879DOI Listing
August 2016