Publications by authors named "Klaus H Metzeler"

76 Publications

NGS-guided precision oncology in metastatic breast and gynecological cancer: first experiences at the CCC Munich LMU.

Arch Gynecol Obstet 2020 Dec 4. Epub 2020 Dec 4.

Department of Obstetrics and Gynecology and CCC Munich LMU University Hospital, Ludwig Maximilians University (LMU), Munich, Germany.

Purpose: Comprehensive genomic profiling identifying actionable molecular alterations aims to enable personalized treatment for cancer patients. The purpose of this analysis was to retrospectively assess the impact of personalized recommendations made by a multidisciplinary tumor board (MTB) on the outcome of patients with breast or gynecological cancers, who had progressed under standard treatment. Here, first experiences of our Comprehensive Cancer Center Molecular Tumor Board are reported.

Methods: All patients were part of a prospective local registry. 95 patients diagnosed with metastatic breast cancer or gynecological malignancies underwent extended molecular profiling. From May 2017 through March 2019, the MTB reviewed all clinical cases considering tumor profile and evaluated molecular alterations regarding further diagnostic and therapeutic recommendations.

Results: 95 patients with metastatic breast or gynecological cancers were discussed in the MTB (68% breast cancer, 20% ovarian cancer, 5% cervical cancer, 3% endometrial cancer and 4% others). Genes with highest mutation rate were PIK3CA and ERBB2. Overall, 34 patients (36%) received a biomarker-based targeted therapy recommendation. Therapeutic recommendations were implemented in nine cases; four patients experienced clinical benefit with a partial response or disease stabilization lasting over 4 months.

Conclusion: In the setting of a multidisciplinary molecular tumor board, a small but clinically meaningful group of breast and gynecological cancer patients benefits from comprehensive genomic profiling. Broad and successful implementation of precision medicine is complicated by patient referral at late stage disease and limited access to targeted agents and early clinical trials.

Trial Registration Number: 284-10 (03.05.2018).
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http://dx.doi.org/10.1007/s00404-020-05881-zDOI Listing
December 2020

An Immune Risk Score Predicts Survival of Patients with Acute Myeloid Leukemia Receiving Chemotherapy.

Clin Cancer Res 2021 Jan 1;27(1):255-266. Epub 2020 Dec 1.

Department of Hematologic Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, P.R. China.

Purpose: Prediction models for acute myeloid leukemia (AML) are useful, but have considerable inaccuracy and imprecision. No current model includes covariates related to immune cells in the AML microenvironment. Here, an immune risk score was explored to predict the survival of patients with AML.

Experimental Design: We evaluated the predictive accuracy of several algorithms for immune composition in AML based on a reference of multi-parameter flow cytometry. CIBERSORTx was chosen to enumerate immune cells from public datasets and develop an immune risk score for survival in a training cohort using least absolute shrinkage and selection operator Cox regression model.

Results: Six flow cytometry-validated immune cell features were informative. The model had high predictive accuracy in the training and four external validation cohorts. Subjects in the training cohort with low scores had prolonged survival compared with subjects with high scores, with 5-year survival rates of 46% versus 19% ( < 0.001). Parallel survival rates in validation cohorts-1, -2, -3, and -4 were 46% versus 6% ( < 0.001), 44% versus 18% ( = 0.041), 44% versus 24% ( = 0.004), and 62% versus 32% ( < 0.001). Gene set enrichment analysis indicated significant enrichment of immune relation pathways in the low-score cohort. In multivariable analyses, high-risk score independently predicted shorter survival with HRs of 1.45 ( = 0.005), 2.12 ( = 0.004), 2.02 ( = 0.034), 1.66 ( = 0.019), and 1.59 ( = 0.001) in the training and validation cohorts, respectively.

Conclusions: Our immune risk score complements current AML prediction models.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3417DOI Listing
January 2021

Identification of BCL-XL as highly active survival factor and promising therapeutic target in colorectal cancer.

Cell Death Dis 2020 10 17;11(10):875. Epub 2020 Oct 17.

Department of Medical Oncology, National Center for Tumor Diseases (NCT), University Hospital Heidelberg, 69120, Heidelberg, Germany.

Since metastatic colorectal cancer (CRC) is a leading cause of cancer-related death, therapeutic approaches overcoming primary and acquired therapy resistance are an urgent medical need. In this study, the efficacy and toxicity of high-affinity inhibitors targeting antiapoptotic BCL-2 proteins (BCL-2, BCL-XL, and MCL-1) were evaluated. By RNA sequencing analysis of a pan-cancer cohort comprising >1500 patients and subsequent prediction of protein activity, BCL-XL was identified as the only antiapoptotic BCL-2 protein that is overactivated in CRC. Consistently, pharmacologic and genetic inhibition of BCL-XL induced apoptosis in human CRC cell lines. In a combined treatment approach, targeting BCL-XL augmented the efficacy of chemotherapy in vitro, in a murine CRC model, and in human ex vivo derived CRC tissue cultures. Collectively, these data show that targeting of BCL-XL is efficient and safe in preclinical CRC models, observations that pave the way for clinical translation.
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http://dx.doi.org/10.1038/s41419-020-03092-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568722PMC
October 2020

Characterization of a Novel FLT3 BiTE Molecule for the Treatment of Acute Myeloid Leukemia.

Mol Cancer Ther 2020 09 9;19(9):1875-1888. Epub 2020 Jun 9.

Amgen Research, Amgen Inc., South San Francisco, California.

Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell-dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines , and FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells , reduced tumor growth and increased survival in AML mouse models Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys , including elimination of FLT3-positive cells in blood and bone marrow. In cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-1093DOI Listing
September 2020

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

Therapeutic management of neuro-oncologic patients - potential relevance of CSF liquid biopsy.

Theranostics 2020 1;10(2):856-866. Epub 2020 Jan 1.

German Cancer Consortium (DKTK), Heidelberg, Germany.

In the era of precision medicine, cancer treatment is increasingly tailored according to tumor-specific genomic alterations. The analysis of tumor-derived circulating nucleic acids in cerebrospinal fluid (CSF) by next generation sequencing (NGS) may facilitate precision medicine in the field of CNS cancer. We therefore evaluated whether NGS from CSF of neuro-oncologic patients reliably detects tumor-specific genomic alterations and whether this may help to guide the management of patients with CNS cancer in clinical practice. : CSF samples from 27 patients with various primary and secondary CNS malignancies were collected and evaluated by NGS using a targeted, amplicon-based NGS-panel (Oncomine Focus Assay). All cases were discussed within the framework of a molecular tumor board at the Comprehensive Cancer Center Munich. : NGS was technically successful in 23/27 patients (85%). Genomic alterations were detectable in 20/27 patients (74%), 11/27 (40%) of which were potentially actionable. After discussion in the MTB, a change of therapeutic management was recommended in 7/27 (26%) of the cases. However, due to rapid clinical progression, only 4/27 (15%) of the patients were treated according to the recommendation. In a subset of patients (6/27, 22%), a high number of mutations of unknown significance suggestive of a high tumor mutational burden (TMB) were detected. NGS from cerebrospinal fluid is feasible in routine clinical practice and yields therapeutically relevant alterations in a large subset of patients. Integration of this approach into a precision cancer medicine program might help to improve therapeutic options for patients with CNS cancer.
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http://dx.doi.org/10.7150/thno.36884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929982PMC
January 2020

The clinical mutatome of core binding factor leukemia.

Leukemia 2020 06 2;34(6):1553-1562. Epub 2020 Jan 2.

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

The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10-15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum ('mutatome') in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.
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http://dx.doi.org/10.1038/s41375-019-0697-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266744PMC
June 2020

The ParaHox gene Cdx4 induces acute erythroid leukemia in mice.

Blood Adv 2019 11;3(22):3729-3739

Institute of Experimental Cancer Research, Comprehensive Cancer Center and University Hospital of Ulm, Ulm, Germany.

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.
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http://dx.doi.org/10.1182/bloodadvances.2019000761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880902PMC
November 2019

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

Nuclear factor of activated T-cells, NFATC1, governs FLT3-driven hematopoietic stem cell transformation and a poor prognosis in AML.

J Hematol Oncol 2019 07 8;12(1):72. Epub 2019 Jul 8.

Department of Hematology, Oncology and Immunology, University Hospital Giessen and Marburg, Campus Marburg, Philipps University Marburg, Marburg, Germany.

Background: Acute myeloid leukemia (AML) patients with a high allelic burden of an internal tandem duplication (ITD)-mutated FMS-like Tyrosine Kinase-3 (FLT3) have a dismal outcome. FLT3 triggers the proliferation of the quiescent hematopoietic stem cell (HSC) pool but fails to directly transform HSCs. While the inflammatory transcription factor nuclear factor of activated T-cells 2 (NFAT2, NFATC1) is overexpressed in AML, it is unknown whether it plays a role in FLT3-induced HSC transformation.

Methods: We generated a triple transgenic mouse model, in which tamoxifen-inducible Cre-recombinase targets expression of a constitutively nuclear transcription factor NFATC1 to FLT3 positive HSC. Emerging genotypes were phenotypically, biochemically, and also transcriptionally characterized using RNA sequencing. We also retrospectively analyzed the overall survival of AML patients with different NFATC1 expression status.

Results: We find that NFATC1 governs FLT3-driven precursor cell expansion and transformation, causing a fully penetrant lethal AML. FLT3/NFATC1-AML is re-transplantable in secondary recipients and shows primary resistance to the FLT3-kinase inhibitor quizartinib. Mechanistically, NFATC1 rewires FLT3-dependent signaling output in HSC, involving augmented K-RAS signaling and a selective de novo recruitment of key HSC-transforming signaling pathways such as the Hedgehog- and WNT/B-Catenin signaling pathways. In human AML, NFATC1 overexpression is associated with poor overall survival.

Conclusions: NFATC1 expression causes FLT3-induced transcriptome changes, which are associated with HSC transformation, quizartinib resistance, and a poor prognosis in AML.
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http://dx.doi.org/10.1186/s13045-019-0765-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615262PMC
July 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

Clonal hematopoiesis and preleukemia-Genetics, biology, and clinical implications.

Genes Chromosomes Cancer 2019 12 16;58(12):828-838. Epub 2019 Apr 16.

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

Myeloid neoplasms including myelodysplastic syndromes and acute myeloid leukemia (AML) originate from hematopoietic stem cells through sequential acquisition of genetic and epigenetic alterations that ultimately cause the disease-specific phenotype of impaired differentiation and increased proliferation. It has become clear that preleukemic clonal hematopoiesis (CH), characterized by an expansion of stem and progenitor cells that carry somatic mutations but are still capable of normal differentiation, can precede the development of clinically overt myeloid neoplasia by many years. CH commonly develops in the aging hematopoietic system, yet progression to myelodysplasia or AML is rare. The discovery that myeloid neoplasms frequently develop from premalignant precursor conditions that are detectable in many healthy individuals has important consequences for the diagnosis, and potentially for the treatment of these disorders. In this review, we summarize the current knowledge on CH as a precursor of myeloid cancers and the implications of CH-related gene mutations in the diagnostic workup of patients with suspected myelodysplastic syndrome. We will discuss the risk of progression associated with CH in healthy persons and in patients undergoing chemotherapy for a non-hematologic cancer, and the significance of CH in autologous and allogeneic stem cell transplantation. Finally, we will review the significance of preleukemic clones in AML and their persistence in patients who achieve a remission after chemotherapeutic treatment.
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http://dx.doi.org/10.1002/gcc.22756DOI Listing
December 2019

Direct modulation of the bone marrow mesenchymal stromal cell compartment by azacitidine enhances healthy hematopoiesis.

Blood Adv 2018 12;2(23):3447-3461

Department of Medicine III, Technische Universität München, Munich, Germany.

Mesenchymal stromal cells (MSCs) are crucial components of the bone marrow (BM) microenvironment essential for regulating self-renewal, survival, and differentiation of hematopoietic stem/progenitor cells (HSPCs) in the stem cell niche. MSCs are functionally altered in myelodysplastic syndromes (MDS) and exhibit an altered methylome compared with MSCs from healthy controls, thus contributing to disease progression. To determine whether MSCs are amenable to epigenetic therapy and if this affects their function, we examined growth, differentiation, and HSPC-supporting capacity of ex vivo-expanded MSCs from MDS patients in comparison with age-matched healthy controls after direct treatment in vitro with the hypomethylating agent azacitidine (AZA). Strikingly, we find that AZA exerts a direct effect on healthy as well as MDS-derived MSCs such that they favor support of healthy over malignant clonal HSPC expansion in coculture experiments. RNA-sequencing analyses of MSCs identified stromal networks regulated by AZA. Notably, these comprise distinct molecular pathways crucial for HSPC support, foremost extracellular matrix molecules (including collagens) and interferon pathway components. Our study demonstrates that the hypomethylating agent AZA exerts its antileukemic activity in part through a direct effect on the HSPC-supporting BM niche and provides proof of concept for the therapeutic potential of epigenetic treatment of diseased MSCs. In addition, our comprehensive data set of AZA-sensitive gene networks represents a valuable framework to guide future development of targeted epigenetic niche therapy in myeloid malignancies such as MDS and acute myeloid leukemia.
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http://dx.doi.org/10.1182/bloodadvances.2018022053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290099PMC
December 2018

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

Bifunctional PD-1 × αCD3 × αCD33 fusion protein reverses adaptive immune escape in acute myeloid leukemia.

Blood 2018 12 1;132(23):2484-2494. Epub 2018 Oct 1.

Department of Medicine III, University Hospital, and.

The CD33-targeting bispecific T-cell engager (BiTE) AMG 330 proved to be highly efficient in mediating cytolysis of acute myeloid leukemia (AML) cells in vitro and in mouse models. Yet, T-cell activation is correlated with upregulation of programmed cell death-ligand 1 (PD-L1) and other inhibitory checkpoints on AML cells that confer adaptive immune resistance. PD-1 and PD-L1 blocking agents may counteract T-cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a bifunctional checkpoint inhibitory T cell-engaging (CiTE) antibody that combines T-cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade. This is accomplished by fusing the extracellular domain of PD-1 (PD-1), which naturally holds a low affinity to PD-L1, to an αCD3.αCD33 BiTE-like scaffold. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1 attachment increases T-cell activation (3.3-fold elevation of interferon-γ) and leads to efficient and highly selective cytotoxicity against CD33PD-L1 cell lines (50% effective concentration = 2.3-26.9 pM) as well as patient-derived AML cells (n = 8). In a murine xenograft model, the CiTE induces complete AML eradication without initial signs of irAEs as measured by body weight loss. We conclude that our molecule preferentially targets AML cells, whereas high-affinity blockers, such as clinically approved anticancer agents, also address PD-L1 non-AML cells. By combining the high efficacy of T-cell engagers with immune checkpoint blockade in a single molecule, we expect to minimize irAEs associated with the systemic application of immune checkpoint inhibitors and suggest high therapeutic potential, particularly for patients with relapsed/ refractory AML.
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http://dx.doi.org/10.1182/blood-2018-05-849802DOI Listing
December 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

Ten-year outcome of patients with acute myeloid leukemia not treated with allogeneic transplantation in first complete remission.

Blood Adv 2018 07;2(13):1645-1650

The Ohio State University Comprehensive Cancer Center, Columbus, OH.

The probability that adult patients with de novo acute myeloid leukemia (AML) receiving intensive chemotherapy in the absence of allogeneic hematopoietic stem cell transplantation (Allo-HCT) in first complete remission (CR1) will be disease-free at 10 years after diagnosis, a long-term surrogate of cure, is unknown. To address this question, we examined 2551 AML patients (1607 aged <60 years, and 944 aged ≥60 years) enrolled in Cancer and Leukemia Group B treatment protocols and the cytogenetics companion protocol 8461 between 1983 and 2004. At 10 years, 267 (16.6%) of patients aged <60 years and 23 (2.4%) of those aged ≥60 years were alive and disease-free. This disease-free AML group consisted predominantly of patients with core-binding factor AML with t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) and those with a normal karyotype. Occurrences of AML beyond 10 years were infrequent and associated with cytogenetic findings different from those at diagnosis. These data provide evidence that the frequency of long-term cure of AML is low among younger and especially older patients in the absence of Allo-HCT in CR1. In older patients not appropriate for Allo-HCT, these data provide further justification for early use of alternative treatments outside of intensive chemotherapy.
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http://dx.doi.org/10.1182/bloodadvances.2017015222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039651PMC
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

Evolution of Cytogenetically Normal Acute Myeloid Leukemia During Therapy and Relapse: An Exome Sequencing Study of 50 Patients.

Clin Cancer Res 2018 04 12;24(7):1716-1726. Epub 2018 Jan 12.

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

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. 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. 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 constituting a mechanism of cytarabine resistance. Low 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 mutations correlated with shorter time to relapse. 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2344DOI Listing
April 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