0000000000050077

AUTHOR

Thomas Kindler

STAT5 is crucial to maintain leukemic stem cells in acute myelogenous leukemias induced by MOZ-TIF2.

Abstract MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essent…

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Targeting components of the alternative NHEJ pathway sensitizes KRAS mutant leukemic cells to chemotherapy.

Abstract Activating KRAS mutations are detected in a substantial number of hematologic malignancies. In a murine T-cell acute lymphoblastic leukemia (T-ALL) model, we previously showed that expression of oncogenic Kras induced a premalignant state accompanied with an arrest in T-cell differentiation and acquisition of somatic Notch1 mutations. These findings prompted us to investigate whether the expression of oncogenic KRAS directly affects DNA damage repair. Applying divergent, but complementary, genetic approaches, we demonstrate that the expression of KRAS mutants is associated with increased expression of DNA ligase 3α, poly(ADP-ribose) polymerase 1 (PARP1), and X-ray repair cross-comp…

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Monitoring of FLT3 Phosphorylation and FLT3 Ligand Levels in Patients with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) Treated with Midostaurin within the AMLSG 16-10 Trial of the German-Austrian Study Group

Abstract Background: Target inhibition of FLT3 by therapy with the recently FDA- and EMA-approved multi-targeted tyrosine kinase inhibitor (TKI) midostaurin can be monitored by plasma inhibitor activity (PIA) analysis by visualizing the level of target-dephosphorylation as previously described. When combining intensive chemotherapy with midostaurin, we have recently shown that the TKI achieves the lowest level of FLT3 phosphorylation (p-FLT3) at the end of the 1st induction cycle, indicating a deep target inhibition. However, sufficient inhibition could not be maintained during subsequent cycles by midostaurin in combination with chemotherapy, but it was reestablished during maintenance the…

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Cross-Inhibition of Interferon-Induced Signals by GM-CSF Through a Block in Stat1 Activation

We investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on biologic signals induced by interferon-alpha (IFN-alpha) and IFN-gamma. In hematopoietic cell lines, IFN-induced signaling was investigated by Western blotting, electrophoretic mobility shift assays (EMSA), flow cytometry, protein-tyrosine phosphatase (PTP) assays, and RT-PCR. GM-CSF inhibited IFN-alpha-induced and IFN-gamma-induced Stat1 tyrosine phosphorylation in a time-dependent manner. EMSA showed that GM-CSF inhibited IFN-alpha-induced and IFN-gamma-induced IFN-gamma activator sequence (GAS) binding activity. As a consequence, IFN-induced transcription of the early response gene, IFN-stimulated…

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CD19 Isoforms Enabling Resistance to CART-19 Immunotherapy Are Expressed in B-ALL Patients at Initial Diagnosis.

Supplemental Digital Content is available in the text.

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Identification of a novel activating mutation (Y842C) within the activation loop of FLT3 in patients with acute myeloid leukemia (AML).

Fms-like tyrosine kinase 3 (FLT3) receptor mutations as internal tandem duplication (ITD) or within the kinase domain are detected in up to 35% of patients with acute myeloid leukemia (AML). N-benzoyl staurosporine (PKC412), a highly effective inhibitor of mutated FLT3 receptors, has significant antileukemic efficacy in patients with FLT3-mutated AML. Mutation screening of FLT3 exon 20 in AML patients (n = 110) revealed 2 patients with a novel mutation (Y842C) within the highly conserved activation loop of FLT3. FLT3-Y842C-transfected 32D cells showed constitutive FLT3 tyrosine phosphorylation and interleukin 3 (IL-3)-independent growth. Treatment with PKC412 led to inhibition of proliferat…

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Monitoring of Minimal Residual Disease (MRD) of DNMT3A Mutations (DNMT3Amut) in Acute Myeloid Leukemia (AML): A Study of the AML Study Group (AMLSG)

![Graphic][1] Background : The DNA methyltransferase 3A ( DNMT3A) is one of the most frequent mutated genes in AML with a hot spot mutation at codon R882 in 80% of the DNMT3A mut cases. In most of the studies DNMT3A mut predicts for poor overall (OS) and relapse-free survival (RFS). Recently, DNMT3A mut have been associated with age-related clonal hematopoiesis, and they have been identified in early preleukemic stem cells. These findings suggest that DNMT3A mut represents an early event in leukemogenesis and may be part of the leukemia founder clone in most AMLs harboring a DNMT3A mut. We thought to address the question whether MRD monitoring in DNMT3A mut patients (pts) can be used for pr…

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Measurable Residual Disease (MRD) Monitoring in Acute Myeloid Leukemia (AML) with t(8;21)(q22;q22.1) RUNX1-RUNX1T1 Identifies Patients at High Risk of Relapse: Results of the AML Study Group (AMLSG)

Background: Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1) resulting in the RUNX1-RUNX1T1 gene fusion is considered favorable in the 2017 genetic risk stratification by the European LeukemiaNet (ELN). After intensive chemotherapy most patients (pts) achieve complete remission (CR), but relapse occurs in about 50% and is associated with poor prognosis. In this AML subgroup monitoring of measurable residual disease (MRD) has been shown to identify pts at higher risk of relapse. Aims: To assess the prognostic impact of MRD monitoring in bone marrow (BM) and peripheral blood (PB) in a large cohort of 155 clinically well-annotated t(8;21)-AML pts enrolled in one of six AMLSG treatment tria…

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Expression Of FLT3-ITD Dysregulates The DBC1-Sirt1-p53 Signaling and Promotes Therapy Resistance

Abstract Background SIRT1 is a NAD+ dependent histone deacetylase, which has been shown to act as an important regulator of apoptosis, DNA-repair and is involved in the maintenance of genetic integrity under conditions of cellular stress. Beside deacetylation of histones H4K16, SIRT1 has numeral other substrates including KU70, FOXO1 or p53. SIRT1 deacetylates p53 at lysine 382 thereby reducing its transcriptional activity followed by loss of p53 dependent apoptosis in response to cell damage. The activity of SIRT1 is negatively regulated by DBC1 (Deleted in Breast Cancer 1) and involves protein–protein interaction (Kim et al., Nature 2008). Recent reports have demonstrated increased expres…

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Activity of Oral Panobinostat (LBH589) in Patients with Myelofibrosis.

Abstract Abstract 2898 Poster Board II-874 Introduction: Panobinostat (LBH589) is a potent pan-deacetylase inhibitor (DACi) targeting epigenetic and non-epigenetic oncogenic pathways. Panobinostat is currently under clinical investigation in a variety of solid tumors and hematologic malignancies. A Phase IA/II trial evaluating oral panobinostat in patients (pts) with advanced hematologic malignancies is currently ongoing, and encouraging clinical activity has been reported previously in pts with lymphoma, myeloma, or leukemia. Here, preliminary activity of oral panobinostat in pts with myelofibrosis (MF) is described. Patients and methods: Pts with advanced hematologic malignancies were tre…

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Impact Of The Pretreatment Characteristics As Well As Cyto- and Molecular-Genetic Profile On Outcome After Relapse In Acute Myeloid Leukemia

Abstract Background Cyto- and molecular-genetic abnormalities evaluated at initial diagnosis are the most powerful prognostic and in part also predictive markers in acute myeloid leukemia (AML) with regard to achievement of complete remission (CR) and survival. Nonetheless, after relapse the prognostic impact of clinical characteristics and genetic abnormalities assessed at initial diagnosis with respect to achievement of subsequent CR and survival are less clear. Aims To evaluate the probability of CR achievement and survival in relapsed AML patients in correlation to clinical characteristics and genetic abnormalities assessed at initial diagnosis as well as treatment strategy. Methods The…

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Updated Results from the German Mpnsg-0212 Combination Trial: Ruxolitinib Plus Pomalidomide in Myelofibrosis with Anemia

Background: Anemia remains one cardinal symptom associated with reduced quality of life (QoL) in patients (pts) with myelofibrosis (MF) which is normally not being addressed by ruxolitinib (RUX). In our previous MPNSG-0109 trial, single-agent pomalidomide (POM) improved cytopenia in 14% (POM 0.5 mg QD) and 29% (POM 2.0 mg QD) of MF pts, respectively. In the MPNSG-0212 study, we sought to investigate the potential synergism of RUX plus POM to improve anemia and QoL in MF pts. Study Design: MPNSG-0212 is an ongoing multicenter, open-label, single-arm phase-Ib/II trial with a target population of 90 pts following a two-stage design (NCT01644110). Pts 1-40 in cohort 1 (co1) were treated with RU…

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SIRT1 prevents genotoxic stress-induced p53 activation in acute myeloid leukemia

SIRT1 is an important regulator of cellular stress response and genomic integrity. Its role in tumorigenesis is controversial. Whereas sirtuin 1 (SIRT1) can act as a tumor suppressor in some solid tumors, increased expression has been demonstrated in many cancers, including hematologic malignancies. In chronic myeloid leukemia, SIRT1 promoted leukemia development, and targeting SIRT1 sensitized chronic myeloid leukemia progenitors to tyrosine kinase inhibitor treatment. In this study, we investigated the role of SIRT1 in acute myeloid leukemia (AML). We show that SIRT1 protein, but not RNA levels, is overexpressed in AML samples harboring activating mutations in signaling pathways. In FMS-l…

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Assessment of Clonal Evolution in 42 AML with NPM1 Mutations by Molecular Characterization of Paired Diagnosis and Relapse Samples

Abstract Abstract 237 Mutations in the nucleophosmin 1 (NPM1) gene represent one of the most frequent gene mutations in acute myeloid leukemia (AML), in particular in cytogenetically normal (CN)-AML. NPM1 mutations (NPM1mut) are considered as an early genetic event in the pathogenesis of AML. To address the role of clonal evolution from diagnosis to relapse in NPM1mut AML, we applied high-resolution genome-wide single nucleotide polymorphism (SNP) array analysis using the Affymetrix 6.0 platform to detect copy number alterations (CNAs) and uniparental disomies (UPDs) in paired samples from 42 patients. In addition, we determined NPM1 and FLT3 [internal tandem duplication (ITD) and tyrosine …

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Minimal Residual Disease Monitoring in Acute Myeloid Leukemia (AML) with Translocation t(8;21)(q22;q22): Results of the AML Study Group (AMLSG)

Abstract Background: Acute myeloid leukemia (AML) with t(8;21)(q22;q22) results in the formation of the RUNX1-RUNX1T1 fusion transcript which can be used to monitor minimal residual disease (MRD) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Early identification of patients (pts) with a high risk of relapse will allow pre-emptive therapy including allogeneic hematopoietic cell transplantation (alloHCT). Recent studies in AML with NPM1 mutation or the CBFB-MYH11 gene fusion revealed that MRD persistence is significantly associated with a high risk of relapse. However, the prognostic impact of MRD assessment in RUNX1-RUNX1T1-positive AML is not well established. A…

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Differential impact of allelic ratio and insertion site in FLT3-ITD-positive AML with respect to allogeneic transplantation.

The objective was to evaluate the prognostic and predictive impact of allelic ratio and insertion site (IS) of internal tandem duplications (ITDs), as well as concurrent gene mutations, with regard to postremission therapy in 323 patients with FLT3-ITD-positive acute myeloid leukemia (AML). Increasing FLT3-ITD allelic ratio (P = .004) and IS in the tyrosine kinase domain 1 (TKD1, P = .06) were associated with low complete remission (CR) rates. After postremission therapy including intensive chemotherapy (n = 121) or autologous hematopoietic stem cell transplantation (HSCT, n = 17), an allelic ratio ≥ 0.51 was associated with an unfavorable relapse-free (RFS, P = .0008) and overall survival …

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Novel 3-Azaindolyl-4-arylmaleimides Exhibiting Potent Antiangiogenic Efficacy, Protein Kinase Inhibition, and Antiproliferative Activity

Tumor growth and metastasis are highly associated with the overexpression of protein kinases (PKs) regulating cell growth, apoptosis resistance, and prolonged cell survival. This study describes novel azaindolyl-maleimides with significant inhibition of PKs, such as VEGFR, FLT-3, and GSK-3β which are related to carcinogenesis. Furthermore, these compounds exhibit high kinase selectivity and potent inhibition of angiogenesis and cell proliferation, offering versatile options in cancer treatment strategies.

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Assessment of Treatment Effects By Measurable Residual Disease Monitoring in NPM1-Mutated AML Patients Randomized for Gemtuzumab-Ozogamicin (GO) within the AMLSG 09-09 Trial of the German-Austrian AML Study Group (AMLSG)

Abstract Background: Measurable residual disease (MRD), as determined by quantitation of Nucleophosmin 1-mutated (NPM1mut) transcript levels (TL), provides significant prognostic information independent of other risk factors in patients (pts) with acute myeloid leukemia (AML). This is also addressed by the 2017 European LeukemiaNet (ELN) risk stratification system, which recommends taking into account results from MRD monitoring when selecting the appropriate post-remission therapy. Furthermore, MRD monitoring provides a powerful tool to evaluate treatment effects within clinical trials investigating novel therapies. Aims: To determine the impact of the anti-CD33 immunotoxin Gemtuzumab-Ozog…

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Sustained complete hematologic remission after administration of the tyrosine kinase inhibitor imatinib mesylate in a patient with refractory, secondary AML.

Abstract Imatinib mesylate, a tyrosine kinase inhibitor targeting bcr-abl, platelet-derived growth factor receptor (PDGF-R), and c-Kit, effectively induces hematologic and cytogenetic remissions in bcr-abl+ chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) with only mild to moderate side effects. Here, we describe the successful treatment of a 64-year-old man with c-Kit+ secondary acute myeloid leukemia (AML) refractory to standard chemotherapy. Upon 2 weeks of imatinib mesylate administration, the patient achieved a complete hematologic remission in peripheral blood. In addition, complete clearance of leukemic blasts in bone marrow and a significant cytogenetic response…

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Sustained Complete Molecular Remissions After Treatment With Imatinib-Mesylate in Patients With Failure After Allogeneic Stem Cell Transplantation for Chronic Myelogenous Leukemia: Results of a Prospective Phase II Open-Label Multicenter Study

Purpose In the era of molecular therapy of chronic myelogenous leukemia (CML) applying BCR-ABL tyrosine kinase inhibitors, the usefulness of molecular end points, in particular, quantitative polymerase chain reaction (PCR) for BCR-ABL in monitoring responses has been broadly accepted. Therefore, we have designed a prospective phase II trial in CML, which, for the first time, evaluated the feasibility and safety of molecular end points as surrogate markers to guide through a stratified treatment algorithm within a multicenter trial. Patients and Methods As a clinical model, we adopted minimal residual disease (MRD) found in relapse after allogeneic stem cell transplantation (SCT) in CML. For…

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Midostaurin in Combination with Intensive Induction and As Single Agent Maintenance Therapy after Consolidation Therapy with Allogeneic Hematopoietic Stem Cell Transplantation or High-Dose Cytarabine (NCT01477606)

Abstract Background: Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in roughly 25% of younger adult patients (pts) with acute myeloid leukemia (AML), implicating FLT3 as a potential target for kinase inhibitor therapy. The multi-targeted kinase inhibitor midostaurin shows potent activity against FLT3 as a single agent but also in combination with intensive chemotherapy. Aims: To evaluate the feasibility and efficacy of midostaurin in combination with intensive induction therapy and as single agent maintenance therapy after allogeneic hematopoietic stem cell transplantation (alloHSCT) or high-dose cytarabine (HIDAC). Methods: The study includes adult pts (age 1…

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Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was foll…

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Genomic Landscape and Molecular Risk in Patients with Advanced Myelofibrosis Treated within the Multicenter Phase Ib/II MPNSG0212 (POMINC) Trial

Abstract Introduction: Mutations (muts) in JAK2, MPL, and CALR are genetic hallmarks in myeloproliferative neoplasms such as myelofibrosis (MF). Prognostication in MF is predominantly based on clinical parameters according to the Dynamic International Prognostic Scoring System (DIPSS). However, gene mutations become increasingly important allowing for a more precised assessment of prognosis. For instance, CALR mutated MF is associated with favorable prognosis, while mutations in distinct high molecular-risk (HMR) genes are considered adverse. Our multicenter phase-Ib/II MPNSG-0212 trial (NCT01644110) investigating ruxolitinib plus pomalidomide in a total cohort of 92 patients with advanced …

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Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia.

Mutations in the nucleophosmin 1 (NPM1) gene are considered a founder event in the pathogenesis of acute myeloid leukemia (AML). To address the role of clonal evolution in relapsed NPM1-mutated (NPM1mut) AML, we applied high-resolution, genome-wide, single-nucleotide polymorphism array profiling to detect copy number alterations (CNAs) and uniparental disomies (UPDs) and performed comprehensive gene mutation screening in 53 paired bone marrow/peripheral blood samples obtained at diagnosis and relapse. At diagnosis, 15 aberrations (CNAs, n = 10; UPDs, n = 5) were identified in 13 patients (25%), whereas at relapse, 56 genomic alterations (CNAs, n = 46; UPDs, n = 10) were detected in 29 patie…

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Clinical Course and Significance of the Novel FLT3-Y842C Mutation in a Patient with AML Treated with PKC412 Monotherapy.

Abstract We recently identified a novel mutation (Y842C) within the tyrosine kinase domain of FLT3 in a patient treated with PKC410 monotherapy (ASH 2003, # 4681). Here, we present follow up studies including the clinical course of the patient and frequency analysis in 110 patients with AML. In addition, we characterized the novel mutation using overexpression of FLT3-Y842C in 32D cells. AML M2 was diagnosed in a 63 year old, male patient in 1993. After having experienced his second relapse upon standard therapy the patient was refractory to alemtuzumab treatment. Due to reduced performance status the patient was not eligible to standard chemotherapy and was enrolled into a phase II trial i…

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Mechanisms of Resistance to the FLT3-Tyrosine Kinase Inhibitor PKC412 in Patients with AML.

Abstract The FLT3 receptor tyrosine kinase is expressed in 70-90% of cases of AML. Up to 35% of patients with AML show mutations in the JM-region or kinase domain of FLT3. These lead to autophosphorylation promoting ligand-independent cell proliferation and inhibition of apoptosis. Treatment with FLT3 tyrosine kinase inhibitors (TKI) is a promising tool in therapy of AML. Preliminary results investigating the FLT3-TKI PKC412 in patients with relapsed/refractory AML revealed that 11/15 patients (73%) with mutated FLT3 and 16/46 patients (35%) with WT FLT3 showed a >50% blast response in peripheral blood (Estey E et al. Blood.2003; 102:919a). Despite its remarkable efficacy in reducing…

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Synergistic targeting of FLT3 mutations in AML via combined menin-MLL and FLT3 inhibition

Abstract The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors…

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Clinical Relevance of Minimal Residual Disease Monitoring in NPM1 Mutated AML: A Study of the AML Study Group (AMLSG)

Abstract Background: Nucleophosmin (NPM1mut) mutations represent one of the most common gene mutations in acute myeloid leukaemia (AML) and can be used for monitoring minimal residual disease (MRD). In a former study, we could define clinical relevant check-points and a cut-off value to identify patients (pts) at high risk of relapse. Aims: To confirm our previous results on the clinical relevance of NPM1mut transcript levels (TL) in an extended cohort of younger AML pts (18 to 60 years) harbouring NPM1mut type A, B, C, D, JT, 4, QM, NM or KM, and to assess the impact of concurrent FLT3 internal tandem duplications (ITD) and DNMT3A (DNMT3Amut) mutations on NPM1mut TL kinetics. Methods: All …

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Next-Generation Sequencing (NGS)-Based Measurable Residual Disease (MRD) Monitoring in Acute Myeloid Leukemia with FLT3 Internal Tandem Duplication (FLT3-ITD+ AML) Treated with Additional Midostaurin

Background: FLT3-ITD occurs in ~25% of adult AML patients (pts) and is associated with poor prognosis. MRD monitoring is of high prognostic relevance, but restricted to certain AML subtypes. FLT3-ITD represents an attractive target for MRD monitoring in particular in pts treated with a tyrosine kinase inhibitor. FLT3-ITD MRD monitoring is hampered by the broad heterogeneity of ITD length and insertion site (IS). NGS may overcome these limitations offering the opportunity for MRD monitoring in FLT3-ITD+ AML. Aims: To validate our recently established NGS-based FLT3-ITD MRD assay in a defined cohort of FLT3-ITD+ AML pts treated within the AMLSG16-10 trial (NCT01477606) combining intensive che…

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Impact of Age and Midostaurin-Dose on Response and Outcome in Acute Myeloid Leukemia with FLT3-ITD: Interim-Analyses of the AMLSG 16-10 Trial

Abstract Background: Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in roughly 25% of younger adult patients (pts) with acute myeloid leukemia (AML). The multi-targeted kinase inhibitor midostaurin combined with intensive chemotherapy has shown activity against AML with FLT3 mutations. However, toxicity and potential drug-drug interactions with strong CYP3A4 inhibitors such as posaconazole may necessitate dose reduction. Aims: To evaluate the impact of age and midostaurin dose-adaptation after intensive induction chemotherapy on response and outcome in AML with FLT3-ITD within the AMLSG 16-10 trial (NCT01477606). Methods: The study included adult pts (age 18-7…

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The JAK2 Kinase Inhibitor LS104 Induces Growth-Arrest and Apoptosis in JAK2V617F Positive Cells.

Abstract The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase i…

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DNMT3A mutations Predict for Inferior Outcome in NPM1-Wildtype and Molecular Unfavorable Cytogenetically-Normal Acute Myeloid Leukemia: A Study of the German-Austrian AMLSG

Abstract Abstract 415 Background: Alteration of DNA methylation, a hallmark of epigenetic modification, is currently discussed as one important pathomechanism in leukemogenesis. Using a next-generation sequencing approach, a frameshift mutation of the gene encoding the DNA methyltransferase (DNMT3A) in an acute myeloid leukemia (AML) case was identified. DNMT3A catalyses the addition of a methyl group to the cytosine residue of CpG dinucleotides, thereby affecting promoter methylation status and gene expression. Subsequent sequencing analysis in an independent cohort of 288 AML patients (pts) revealed DNMT3A mutations (DNMT3Amut) in 22% of the pts; mutations were associated with intermediat…

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Combined Targeting of the Menin-MLL1 Chromatin Complex and FLT3 As a Novel Therapeutic Concept Against NPM1 Mutant or MLL-Rearranged AML with Mutated FLT3

NPM1mutant (NPM1mut) and MLL1-rearranged (MLL-r) acute myeloid leukemias (AMLs) exhibit aberrant expression of HOX and MEIS1 transcription factors and commonly harbor an activating mutation in the receptor tyrosine kinase FLT3. Pharmacologic inhibition of the menin-MLL1 complex reverses leukemogenic gene expression including MEIS1 and FLT3 and represents a therapeutic opportunity for the treatment of these leukemias. Here, we investigate the contribution of the menin-MLL1 complex to leukemic FLT3 signaling and assess the therapeutic potential of dual menin-MLL1 and FLT3 targeting. First, we performed RNA sequencing to delineate transcriptional changes associated with menin-MLL1 inhibition (…

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Abstract LB-287: Identification of patients at risk for tumor predisposition syndromes based on the evaluation of sporadic cancer exome sequencing data: experiences from the NCT/DKTK MASTER program

Abstract The MASTER (Molecularly Aided Stratification for Tumor Eradication Research) Program of the NCT (National Center for Tumor Diseases) Heidelberg and the DKTK (German Cancer Consortium) is situated at the interface of cancer genomics and clinical oncology to provide whole exome/genome and transcriptome sequencing to selected patients with unmet medical need, and to evaluate the utility of such an approach regarding molecular stratification and individualized, biology-guided treatment. The program has enabled implementation of a shared, DKTK-wide workflow for rapid-turnaround clinical sequencing, comprising all steps from sample processing to reporting of results by a dedicated molecu…

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Targeting Aberrant Ncam (neural cell adhesion molecule; CD56) Expression in Acute Myeloid Leukemia

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease of the hematopoietic progenitor cell driven by the subsequent acquisition of genetic alterations. Approximately 20% of AML patients show strong expression of CD56 (neural cell adhesion molecule; NCAM). Expression of NCAM is associated with poor overall survival; however, the functional role of aberrant NCAM expression has not been investigated to date. The goal of this study is to examine the biological role of NCAM in AML and to explore whether NCAM represents a potential therapeutic target. Results In order to evaluate the clinical significance of elevated NCAM expression in AML, we screened a panel of human cell …

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Paraoxonase-2 alters hematopoietic stem cell differentiation through redox signalling

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An Adapted Gating Strategy Integrating a Myelomonocytic Window Is Necessary For Correct Flow Cytometric Diagnosis In a Large Proportion Of AML With Mutated NPM1

Abstract Background Previously, it has been reported, that AML with mutated NPM1 is associated with a distinctive immunophenotype. In particular, low or absent expression of CD34 accompanied by high expression of CD33, and – at least in part of the cases - absence of HLA-DR expression was reported. CD45/side scatter (SSC) gating is widely used for the identification of blasts by flow cytometry (FC). Blast cell gates typically are defined by a low SSC and moderate CD45 expression. However, in a number of patients with NPM1mutation this typical blast cell gate comprises significantly lower blast percentages when compared to the morphological evaluation. In these patient samples a second popul…

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Phase ia/ii, two-arm, open-label, dose-escalation study of oral panobinostat administered via two dosing schedules in patients with advanced hematologic malignancies

Panobinostat is a potent oral pandeacetylase inhibitor that leads to acetylation of intracellular proteins, inhibits cellular proliferation and induces apoptosis in leukemic cell lines. A phase Ia/II study was designed to determine the maximum-tolerated dose (MTD) of daily panobinostat, administered on two schedules: three times a week every week or every other week on a 28-day treatment cycle in patients with advanced hematologic malignancies. The criteria for hematologic dose-limiting toxicities differed between patients with indications associated with severe cytopenias at baseline (leukemia and myeloid disorders) and those less commonly associated with baseline cytopenias (lymphoma and …

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Drivers of topoisomerase II poisoning mimic and complement cytotoxicity in AML cells

Recently approved cancer drugs remain out-of-reach to most patients due to prohibitive costs and only few produce clinically meaningful benefits. An untapped alternative is to enhance the efficacy and safety of existing cancer drugs. We hypothesized that the response to topoisomerase II poisons, a very successful group of cancer drugs, can be improved by considering treatment-associated transcript levels. To this end, we analyzed transcriptomes from Acute Myeloid Leukemia (AML) cell lines treated with the topoisomerase II poison etoposide. Using complementary criteria of co-regulation within networks and of essentiality for cell survival, we identified and functionally confirmed 11 druggabl…

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In BCR-ABL-positive cells, STAT-5 tyrosine-phosphorylation integrates signals induced by imatinib mesylate and Ara-C.

In BCR-ABL-positive cells, the transcription factor STAT-5 is constitutively activated by tyrosine phosphorylation. STAT-5 activation results in upregulation of bcl-X(L) and increased resistance to induction of apoptosis. Here, we investigated the effects of imatinib mesylate and cytosine arabinoside (Ara-C) on STAT-5 tyrosine-phosphorylation, cellular proliferation and induction of apoptosis in cell lines and primary hematopoietic cells. Imatinib mesylate treatment strongly suppressed STAT-5 tyrosine-phosphorylation in K562 and primary CML blasts. In contrast to JAK-2 and PI-3-kinase inhibition, exposure of K562 cells to imatinib mesylate resulted in obvious suppression of proliferation. R…

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A phase II study of elacytarabine in combination with idarubicin and of human equilibrative nucleoside transporter 1 expression in patients with acute myeloid leukemia and persistent blasts after the first induction course.

Unlike cytarabine, cellular entry of Elacytarabine, the elaidic acid ester derivative of cytarabine, is independent of the human equilibrative nucleoside transporter 1 (hENT1). This phase II study tested whether the hENT1 blast expression level can be used as a predictive marker for cytarabine response and if the efficacy of elacytarabine is independent of hENT1 expression. A total of 51 patients with acute myeloid leukemia (AML) induction failure were given elacytarabine-idarubicin as a second induction course. The hENT1 expression level was analyzed prior to first induction and/or prior to treatment with elacytarabine. The overall response rate (ORR) was 41% and the safety profile was man…

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Validating comprehensive next-generation sequencing results for precision oncology : The NCT/DKTK molecularly aided stratification for tumor eradication research experience

Purpose Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameter…

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Clinical Impact of GATA2 Mutations in Acute Myeloid Leukemia Patients Harboring CEBPA Mutations: A Study of the AML Study Group (AMLSG)

Abstract Background Based on their association with certain biological and clinical features as well as their prognostic significance, mutations in the CCAAT/enhancer-binding protein-alpha (CEBPA) gene have been included as a provisional entity into the 2008 World Health Organization (WHO) classification of myeloid neoplasms. CEBPA mutations (CEBPAmut) are mainly found in acute myeloid leukemia (AML) with normal cytogenetics, and approximately 60% of the mutated patients (pts) carry biallelic mutations. Several studies showed that in particular pts with double mutant CEBPA (CEBPAdm) have a favorable outcome compared to all others. Recently, mutations in the transcription factor GATA2 were i…

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Comprehensive Genomic and Transcriptomic Analysis for Guiding Therapeutic Decisions in Patients with Rare Cancers

Abstract The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patient…

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Efficacy and safety of imatinib in adult patients with c-kit–positive acute myeloid leukemia

Abstract This phase 2 pilot study was conducted to determine the efficacy and safety of imatinib mesylate in patients with c-kit–positive acute myeloid leukemia (AML) refractory to or not eligible for chemotherapy. Twenty-one patients were enrolled and received imatinib 600 mg orally once daily. Five responses were seen primarily in patients, starting with relatively low blast counts in bone marrow (BM) and peripheral blood (PB): 2 patients who were considered refractory on chemotherapy on the basis of persistence of blasts in PB and BM met the criteria for complete hematologic remission, 1 patient had no evidence of leukemia, and 2 patients achieved a minor response. Treatment with imatini…

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Analysis of Differentially Activated Signaling Pathways in Myeloproliferative Disease Using Kinomics Chip Profiling

Abstract In a multitude of cases, oncogenic mutations are gain of function mutations that confer a constitutively activated gene product. Currently, evidence from a large body of experimental studies suggests that oncogenic transformation induced by activating kinase mutations is not sufficiently explained by constitutive kinase activation alone but is a result of aberrantly activated signaling pathways in affected cells. The JAK2V617F-mutation is a highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, Ba/F3-cells expressing both erythropoietin receptor (EpoR) and the JAK2V617F-mutation show constitutive activation of the JAK-STAT pathway and cytokine …

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Distinct immune evasion in APOBEC-enriched, HPV-negative HNSCC

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (N = 496) and a validation set (DKTK MASTER cohort, N = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately re-analyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-nega…

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LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells

Abstract The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of …

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Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain.

Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an …

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Definitive evidence for Club cells as progenitors for mutantKras/Trp53‐deficient lung cancer

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide de…

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Bioassays to monitor taspase1 function for the identification of pharmacogenetic inhibitors

Background Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm…

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FLT3 as a therapeutic target in AML: still challenging after all these years

Abstract Mutations within the FMS-like tyrosine kinase 3 (FLT3) gene on chromosome 13q12 have been detected in up to 35% of acute myeloid leukemia (AML) patients and represent one of the most frequently identified genetic alterations in AML. Over the last years, FLT3 has emerged as a promising molecular target in therapy of AML. Here, we review results of clinical trials and of correlative laboratory studies using small molecule FLT3 tyrosine kinase inhibitors (TKIs) in AML patients. We also review mechanisms of primary and secondary drug resistance to FLT3-TKI, and from the data currently available we summarize lessons learned from FLT3-TKI monotherapy. Finally, for using FLT3 as a molecul…

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Minimal Residual Disease (MRD) Monitoring in NPM1 Mutated Acute Myeloid Leukemia (AML): Impact of Concurrent FLT3-ITD and DNMT3A Mutations on MRD Kinetics and Clinical Outcome

Abstract Introduction In a recent update on MRD monitoring in 407 NPM1 mutated (NPM1mut) AML patients (pts) we could confirm the results from our previous study showing that achievement of RQ-PCR negativity after double induction (DI), after completion of therapy (CT) as well as during the follow-up period (FUP) is significantly associated with a lower cumulative incidence of relapse (CIR) and superior overall survival (OS) [Döhner K, Annals of Hematol; 2013;Suppl.1,92:S39]. In addition, in pts with concurrent FLT3-ITD (FLT3-ITDmut) or DNMT3A (DNMT3Amut) mutations, we also showed that the median NPM1mut transcript levels after each treatment cycle were significantly higher. Aim To evaluate …

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A phase 1b, dose-finding study of ruxolitinib plus panobinostat in patients with myelofibrosis.

7022^ Background: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by dysregulation of the Janus kinase (JAK) pathway resulting in bone marrow fibrosis, splenomegaly, and debilitat...

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The kinase inhibitor LS104 induces apoptosis, enhances cytotoxic effects of chemotherapeutic drugs and is targeting the receptor tyrosine kinase FLT3 in acute myeloid leukemia.

Activating mutations of FLT3 are found in approximately one-third of acute myeloid leukemia (AML)-cases and are considered to represent an attractive therapeutic target. In this study, we report that the hydroxystyryl-acrylonitrile compound LS104 inhibits proliferation and induces potent cytotoxic effects in FLT3 expressing leukemic cells in vitro. Immunoblot and phosphoprotein-FACS analysis demonstrated inhibiton of phosphorylation of FLT3-ITD and of its downstream targets. In pharmacokinetic studies, a rapid and dose dependent cellular uptake of LS104 lasting up to 11h could be demonstrated. Combination of LS104 with chemotherapeutic agents markedly enhanced cytotoxic effects. Recently, a…

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Efficacy, Safety, and Confirmation of the Recommended Phase 2 Starting Dose of the Combination of Ruxolitinib (RUX) and Panobinostat (PAN) in Patients (Pts) with Myelofibrosis (MF)

Abstract BACKGROUND: MF is a myeloproliferative neoplasm characterized by bone marrow (BM) fibrosis, splenomegaly, and debilitating constitutional symptoms. RUX is a potent JAK1/JAK2 inhibitor that has demonstrated superiority in spleen volume reduction, symptom improvement, and survival in the phase 3 COMFORT studies compared with placebo and best available therapy. PAN, a potent pan-deacetylase inhibitor, inhibits JAK signaling by disrupting the interaction between JAK2 and heat shock protein 90, a protein chaperone. PAN has demonstrated reductions in splenomegaly and improvement of BM fibrosis in phase 1/2 studies. The combination of RUX and PAN demonstrated synergistic activity in precl…

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Exploiting Gangliosides for the Therapy of Ewing’s Sarcoma and H3K27M-Mutant Diffuse Midline Glioma

Simple Summary Osteosarcoma, Ewing’s sarcoma, and H3K27M-mutant diffuse midline glioma are rare but aggressive malignancies occurring mainly in children. Due to their rareness and often fatal course, drug development is challenging. Here, we repurposed the existing drugs dinutuximab and eliglustat and investigated their potential to directly target or indirectly modulate the tumor cell-specific ganglioside GD2. Our data suggest that targeting and/or modulating tumor cell-specific GD2 may offer a new therapeutic strategy for the above mentioned tumor entities. Abstract The ganglioside GD2 is an important target in childhood cancer. Nevertheless, the only therapy targeting GD2 that is approve…

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LS104, a Novel Kinase Inhibitor, Induces Apoptosis, Synergizes with Cytostatic Drugs and Is Targeting the Receptor Tyrosine Kinase FLT3.

Abstract Fms-like tyrosine kinase 3 (FLT3), a member of the class III tyrosine kinase receptor family, is expressed in up to 90% of acute myeloid leukemia (AML). Activating mutations like internal tandem duplication (ITD) of the juxtamembrane domain and kinase domain point mutations are found in approximately 35% of AML-cases and are considered to represent an attractive therapeutic target. In this study, we report that the novel hydroxystyryl-acrylonitrile compound LS104 induces potent cytotoxic effects in FLT3 ITD-positive leukemic cells. As a cellular model to investigate FLT3-ITD specific effects we used 32D myeloid cells stably transfected with FLT3-ITD and wt-FLT3, respectively. In MT…

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Molecular Characterization of Relapsed Core-Binding Factor (CBF) Acute Myeloid Leukemia (AML)

Abstract Background: CBF-AML is defined by recurrent genetic abnormalities which encompass t(8;21)(q22;q22), inv(16)(p13.1q22) or less frequently t(16;16)(p13.1;q22). Most frequent secondary chromosome aberrations in t(8;21) AML are del(9q) or loss of a sex chromosome, and in inv(16)/t(16;16) AML trisomy 22 or trisomy 8. At the molecular level mutations involving KIT, FLT3, or NRAS were identified as recurrent lesions in CBF-AML. However, the underlying genetic alterations which might trigger relapse in CBF-AML are not well delineated. Thus, the aim of our study was to characterize the clonal architecture of relapsed CBF-AML. Methods: We performed mutational profiling (KIT, FLT3-ITD, FLT3-T…

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Distinct immune evasion in APOBEC ‐enriched, HPV ‐negative HNSCC

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negat…

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Efficacy and safety of bosutinib (BOS) for Philadelphia chromosome–positive (Ph+) leukemia in older versus younger patients (pts).

6511 Background: BOS is an oral dual Src/Abl kinase inhibitor with potent activity in Ph+ leukemia. Methods: Efficacy and safety of BOS 500 mg/d was evaluated in older (≥65 y; n = 119) and younger (<65 y; n = 451) pts in 3 cohorts: chronic phase chronic myeloid leukemia (CP CML) after imatinib (IM; CP2L cohort; n = 287); CP CML after IM + dasatinib (DAS) and/or nilotinib (NIL; CP3L cohort; n = 119); and accelerated/blast phase (AP/BP) CML or acute lymphoblastic leukemia after IM ± DAS and/or NIL (ADV cohort; n = 164). Results: Baseline events (≥65 y vs <65 y) included respiratory disorders (35% vs 13%), cardiac disorders (29% vs 9%), and diabetes (4% vs 4%). Median baseline medicatio…

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A novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in AML

Abstract Currently, FLT3 tyrosine kinase inhibitors (TKIs) are emerging as the most promising drug therapy to overcome the dismal prognosis of acute myelogenous leukemia (AML) patients harboring internal tandem duplications (ITDs) of FLT3. However, up-front drug resistance occurs in approximately 30% of patients, and molecular mechanisms of resistance are poorly understood. Here, we have uncovered a novel mechanism of primary resistance to FLT3 TKIs in AML: an FLT3 receptor harboring a nonjuxtamembrane ITD atypically integrating into the β-2 sheet of the first kinase domain (FLT3_ITD627E) induces dramatic up-regulation of the anti-apoptotic myeloid cell leukemia 1 protein (MCL-1). Using RNA…

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A Phase II Study of Elacytarabine/Idarubicin As Second Course Remission-Induction in Patients with Acute Myeloid Leukemia Who Failed Cytarabine/Anthracycline

Abstract Abstract 46 Background Elacytarabine, a fatty acid derivative (elaidic acid ester) of cytarabine, has pharmacokinetic and pharmacodynamic properties that may lead to improved clinical outcomes (Adema et al., 2011) compared to cytarabine. A phase I study had established 1000 mg/m2/d continuous infusion on d 1 – 5 as a safe and effective dose of elacytarabine given in combination with idarubicin at 12 mg/m2/d IV d 1 – 3 (Giles et al., 2012). Mechanism of Action The mechanism of action is similar to cytarabine, but unlike cytarabine, plasma elimination half-life is long, intracellular distribution is prolonged and activity is independent of membrane nucleoside transporters. Resistance…

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Conceptual framework for precision cancer medicine in Germany: Consensus statement of the Deutsche Krebshilfe working group ‘Molecular Diagnostics and Therapy’

Precision cancer medicine (PCM) holds great promises to offer more effective therapies to patients based on molecular profiling of their individual tumours. Although the PCM approach seems intuitive, multiple conceptional and structural challenges interfere with the broad implementation of PCM into clinical practice. Accordingly, concerted national and international efforts are needed to guide the further development and broad adoption of PCM in Germany. With support of the 'German Cancer Aid' (Deutsche Krebshilfe [DKH]) a task force 'Molecular Diagnostics and Therapy' was implemented. In two workshops supported by the DKH, delegates from the fourteen comprehensive cancer centresidentified …

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Condensed Versus Standard Schedule of High-Dose Cytarabine Consolidation Therapy with Pegfilgrastim Growth Factor Support in Acute Myeloid Leukemia

Abstract Background: The concept of intensive post-remission chemotherapy in acute myeloid leukemia (AML) is based on the observation that despite achievement of a first complete remission (CR) after intensive induction therapy virtually all patients relapse in the absence of further treatment. Moreover, randomized studies showed that intensive post-remission consolidation chemotherapy was superior to prolonged low-dose maintenance therapy in younger patients. With regard to consolidation therapy, the landmark study conducted by the Cancer and Leukemia Group B established the current standard for patients aged 60 years and younger with high-dose cytarabine (HDAC) 3g/m² bidaily on days days …

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Durable Molecular Remissions in Patients with Relapsed CML Post Allogeneic Stem Cell Transplantation upon Treatment with Imatinib-Mesylate (Glivec®, STI-571). Follow-Up Results of a Phase II Open-Label Study.

Abstract Purpose: In a phase II clinical trial we have previously reported on the safety and efficacy of imatinib mesylate (IM) to induce hematologic, cytogenetic and molecular remissions in case of relapse post allogeneic stem cell transplantation (SCT) in patients with chronic myelogenous leukaemia (CML). Here we report on an extended follow-up phase, which was performed to monitor stability of responses and further disease course in patients enrolled. Patients and Methods: Within the trial, patients, transplanted in chronic phase (CP) CML with molecular or cytogenetic relapse (n=37), received IM at a starting dose of 400mg. Close monitoring was performed, which, besides evaluation of sid…

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Efficacy, Safety, and Confirmation of the Recommended Phase 2 Dose of Ruxolitinib Plus Panobinostat in Patients with Intermediate or High-Risk Myelofibrosis

Abstract Background: Myelofibrosis (MF) is a clonal neoplastic disease resulting in bone marrow fibrosis, splenomegaly, and debilitating constitutional symptoms. The Janus kinase (JAK) pathway is often dysregulated in MF, and agents targeting this pathway have demonstrated efficacy in this disease. Ruxolitinib (RUX), a potent JAK1/JAK2 inhibitor, demonstrated superiority in spleen volume reduction, symptom improvement, and survival compared with the control arm in the phase III COMFORT-I and COMFORT-II studies. Panobinostat (PAN), a potent pan-deacetylase inhibitor (pan-DACi), inhibits JAK signaling through disruption of the interaction of JAK2 with the protein chaperone heat shock protein …

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Pharmacodynamic Monitoring of the Efficacy of a Targeted Therapy with Midostaurin By Plasma Inhibitor Activity (PIA) Analysis in FLT3 -ITD Positive AML Patients within the AMLSG 16-10 Trial: A Study of the AML Study Group (AMLSG)

Abstract Background: Activating mutations in receptor tyrosine kinases like FLT3 (FLT3mut) lead to an aberrant signal transduction thereby causing an increased proliferation of hematopoietic cells. Internal tandem duplications (FLT3-ITD) or mutations in the tyrosine kinase domain (FLT3-TKD) occur in about 25% of younger adult patients (pts) with acute myeloid leukemia (AML), with FLT3 -ITD being associated with an unfavourable outcome. FLT3mut present an excellent target for small molecule tyrosine kinase inhibitors (TKI). The multi-targeted kinase inhibitor midostaurin (PKC412) is currently under investigation as a FLT3-inhibitor in combination with intensive chemotherapy. Monitoring of th…

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All-Trans Retinoic Acid Improves Outcome in Younger Adult Patients with Nucleophosmin-1 Mutated Acute Myeloid Leukemia – Results of the AMLSG 07-04 Randomized Treatment Trial

Abstract Abstract 80 Background: Mutations in the nucleophosmin-1 gene (NPM1) are the most common genetic abnormalities in acute myeloid leukemia (AML) and define a provisional AML entity in the current WHO classification. In a retrospective biomarker study within a randomized trial of older patients with AML, we demonstrated that patients with mutated NPM1 and absence of a FLT3 internal tandem duplication (ITD) benefit from all-trans retinoic acid (ATRA) as adjunct to conventional chemotherapy (Schlenk et al. Haematologica 2009;94:54–69). Aims: To evaluate the impact of ATRA in combination with conventional chemotherapy on outcome, and to assess the NPM1 mutational status as predictive mar…

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Clinical impact of GATA2 mutations in acute myeloid leukemia patients harboring CEBPA mutations: a study of the AML study group.

Clinical impact of GATA2 mutations in acute myeloid leukemia patients harboring CEBPA mutations: a study of the AML study group

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Ruxolitinib Plus Pomalidomide in Myelofibrosis: Updated Results from the Mpnsg-0212 Trial (NCT01644110)

Abstract Background: Although ruxolitinib (RUX) reduces constitutional symptoms and splenomegaly in myelofibrosis (MF) therapy options for anemia are limited. In our prior MPNSG-0109 trial of pomalidomide (POM) in MF with cytopenia, anemia responses were reported in 14% and 29% of subjects receiving POM 0.5 mg/d and 2 mg/d, respectively (Schlenk RF et al., ASH 2013, abstract #2822). We designed a phase-Ib/-II combination study of RUX plus POM to evaluate synergistic effects in subjects with anemia and splenomegaly (MPNSG-0212 trial, NCT01644110; Stegelmann et al., ASH 2015, abstract #826). Study Design: Primary endpoints are response rate after 12 treatment cycles (28 days each) according t…

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Intracellular retention of ABL kinase inhibitors determines commitment to apoptosis in CML cells

PLoS one 7(7), e40853 (2012). doi:10.1371/journal.pone.0040853

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Different FLT3-ITD Integration Sites Are Associated with Differential Sensitivity to Tyrosine Kinase Inhibitors (TKI) In Vitro.

Abstract Abstract 1709 Introduction: Currently, small molecule FLT3 tyrosine kinase inhibitors (TKIs) are promising therapeutic approaches to overcome the dismal prognosis of AML patients harbouring FLT3-ITD mutations. However, up to 30% of these patients show primary resistance to FLT3-TKIs. Recently, we uncovered a novel mechanism of primary resistance to FLT3 TKIs in a patient displaying an atypical integration site of ITD within the beta2-sheet (ITD_A627E). The data suggested that atypical integration sites of ITDs within the tyrosine kinase domain-1 (TKD1) of FLT3 (beta1-sheet, nucleotide binding loop and beta2-sheet) are associated with rewired signaling and differential responsivenes…

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AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein α (c/EBPα) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D ce…

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A Phase 1b, Dose-Finding Study Of Ruxolitinib Plus Panobinostat In Patients With Primary Myelofibrosis (PMF), Post–Polycythemia Vera MF (PPV-MF), Or Post–Essential Thrombocythemia MF (PET-MF): Identification Of The Recommended Phase 2 Dose

Abstract Background Myelofibrosis (MF) is a myeloproliferative neoplasm associated with progressive, debilitating symptoms that impact patient quality of life (QoL) and reduce survival. Ruxolitinib (RUX), a potent dual JAK1/JAK2 inhibitor, demonstrated superiority in spleen volume and symptom reduction, improved health-related QoL measures, and prolonged survival compared with traditional therapies or placebo in the phase 3 COMFORT studies. Panobinostat (PAN) is a potent oral pan-deacetylase inhibitor (DACi) that inhibits JAK pathway signaling through increased acetylation of the JAK2 protein chaperone HSP90. In phase 1/2 studies in MF, PAN has shown reduction in splenomegaly and JAK2 V617F…

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