Katharina Wagner

Prognostic significance of expression levels of stem cell regulators MSI2 and NUMB in acute myeloid leukemia

Deregulation of the hematopoietic stem cell (HSC) compartment represents a hallmark of acute myeloid leukemia (AML). Recently, in vivo screening for genes that are involved in the regulation of HSCs has led to the discovery of Musashi-2 (MSI2) as a key regulator of HSCs and as a suppressor of NUMB. In order to analyze the prognostic importance of MSI2 and NUMB expression in AML, MSI2 and NUMB transcript levels from 454 AML patients treated in multicenter trials AML SHG 0199 (ClinicalTrials Identifier NCT00209833) and 0295, and 38 healthy volunteers were analyzed by reverse transcriptase PCR in the context of other molecular markers (NPM1, FLT3, CEBPA, IDH1/IDH2, DNMT3A, NRAS, WT1, KIT, MN1, BAALC, ERG, and WT1). In AML, patients with high MSI2 expression were more likely to be FLT3-ITD positive (Pu2009<u2009.001), NPM1 (Pu2009<u2009.001), and DNMT3A (Pu2009=u2009.003) mutated. Overall survival (OS) was shorter in AML patients with high MSI2 expression (hazard ratio, 1.48; 95xa0% confidence interval, 1.13–1.95, Pu2009=u2009.005). However, relapse-free survival (RFS, Pu2009=u2009.15) and complete remission (CR, Pu2009=u2009.39) rates were not influenced by MSI2 expression. In multivariate analysis, MSI2 expression remained an independent prognostic factor for OS (Pu2009=u2009.03). NUMB expression had no impact on survival (OS, Pu2009=u2009.47; RFS, Pu2009=u2009.59) and CR rate (Pu2009=u2009.39). MSI2 but not NUMB is associated with shorter OS in AML patients and may indicate a more aggressive form of AML.

Analysis of NUP98/NSD1 translocations in adult AML and MDS patients

S Anguille, F Fujiki, EL Smits, Y Oji, E Lion, Y Oka, ZN Berneman and H Sugiyama Center for Cell Therapy & Regenerative Medicine, Antwerp University Hospital, Edegem, Antwerp, Belgium; Vaccine & Infectious Disease Institute (VAXINFECTIO), Laboratory of Experimental Hematolog, University of Antwerp, Wilrijk, Antwerp, Belgium; Department of Cancer Immunology, Osaka University Graduate School of Medicine, Osaka, Japan; Department of Cancer Stem Cell Biology, Osaka, Japan; Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka, Japan and Department of Functional Diagnostic Science, Osaka, Japan E-mail: sebastien.anguille@uza.be These authors contributed equally to this work.

Expression of Hedgehog Pathway Mediator GLI Represents a Negative Prognostic Marker in Human Acute Myeloid Leukemia and Its Inhibition Exerts Antileukemic Effects

Purpose: The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML). Experimental Design: Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo. Results: Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice. Conclusions: GLI expression is a negative prognostic factor and might represent a novel druggable target in AML. Clin Cancer Res; 21(10); 2388–98. ©2015 AACR.

Prognostic significance of combined MN1, ERG, BAALC, and EVI1 (MEBE) expression in patients with myelodysplastic syndromes

Overexpression of MN1, ERG, BAALC, and EVI1 (MEBE) genes in cytogenetically normal acute myeloid leukemia (AML) patients is associated with poor prognosis, but their prognostic effect in patients with myelodysplastic syndromes (MDS) has not been studied systematically. Expression data of the four genes from 140 MDS patients were combined in an additive score, which was validated in an independent patient cohort of 110 MDS patients. A high MEBE score, defined as high expression of at least two of the four genes, predicted a significantly shorter overall survival (OS) (HR 2.29, 95xa0% CI 1.3–4.09, Pu2009=u2009.005) and time to AML progression (HR 4.83, 95xa0% CI 2.01–11.57, Pu2009<u2009.001) compared to a low MEBE score in multivariate analysis independent of karyotype, percentage of bone marrow blasts, transfusion dependence, ASXL1, and IDH1 mutation status. In a validation cohort of 110 MDS patients, a high MEBE score predicted shorter OS (HR 1.77; 95xa0% CI 1.04–3.0, Pu2009=u2009.034) and time to AML progression (HR 3.0, 95xa0% CI 1.17–7.65, Pu2009=u2009.022). A high MEBE expression score is an unfavorable prognostic marker in MDS and is associated with an increased risk for progression to AML. Expression of the MEBE genes is regulated by FLI1 and c-MYC, which are potential upstream targets of the MEBE signature.

Functional Analysis of a Single Chain Chimeric α/β-Granulocyte-Macrophage Colony-stimulating Factor Receptor IMPORTANCE OF A GLUTAMATE RESIDUE IN THE TRANSMEMBRANE REGION

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the α-subunit (α-GMR) to the intracellular part of the β-subunit (β-GMR) introducing an additional glutamate residue at the fusion site (α/β-GMR). We demonstrated the capacity of α/β-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this α/β-GMR is sufficient to mediate morphological changes, expression of α4- and β1-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of α/β-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when β-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not α/β-GMR can induce proliferation in the presence of an anti-α-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of α/β-GMR for ligand-induced receptor activation.

Lack of noncanonical RAS mutations in cytogenetically normal acute myeloid leukemia

Transforming mutations in RAS genes are commonly found in human malignancies, including myeloid leukemias. To investigate the incidence, spectrum, and distribution of activating K- and N-RAS mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients, 204 CN-AML patients were screened. Activating K- and N-RAS mutations were detected in 3 of 204 (1.5xa0%) and 22 of 204 (10.8xa0%) CN-AML samples, respectively. RAS mutated patients presented with a lower percentage of bone marrow blasts (65 vs 80xa0%, Pu2009=u20090.022). RAS mutations tended to occur with nucleophosmin-1 (NPM1) mutations (Pu2009=u20090.079), and all three samples containing K-RAS mutations had concomitant NPM1 mutations. There was no significant overlap between K-RAS mutations and N-RAS, FLT3, CEBPA, IDH1/2, WT1 or MLL mutations. RAS mutation status did not impact relapse-free or overall survival of CN-AML patients. In contrast to reports of noncanonical RAS mutations in other cancers, including some leukemia subtypes, we only observed K- and N-RAS mutations in codons 12, 13, or 61 in CN-AML samples. Our findings suggest that while K-RAS mutations are infrequent in CN-AML, activating K-RAS mutations may cooperate with mutated NPM1 to induce leukemia.

ID1 expression associates with other molecular markers and is not an independent prognostic factor in cytogenetically normal acute myeloid leukaemia.

In acute myeloid leukaemia with normal karyotype (CN‐AML), gene mutations (e.g. NPM1, FLT3, CEBPA) as well as deregulated gene expression affect outcome. High expression of ID1 was described as a negative prognostic factor. We have shown that CEBPA regulates ID1 expression. Therefore, we analysed the prognostic impact of ID1 expression in 269 patients (aged 16–60 years) with CN‐AML in the context of other molecular markers, particularly CEBPA mutations. ID1high status was an independent negative prognostic factor for overall survival (OS) in multivariate analysis when analysed together with age, extramedullary disease, platelets, expression of BAALC and WT1, FLT3‐internal tandem duplication, NPM1, WT1 single nucleotide polymorphism rs16754 and IDH1. ID1 expression was higher in CEBPA wildtype patients than in patients with monoallelic CEBPA mutations and these patients showed higher ID1 expression compared to patients with biallelic CEBPA mutations. Thus, when CEBPA mutations were considered, ID1 expression lost its prognostic impact. Likewise, the negative impact of ID1high status on relapse‐free survival (RFS) was lost when CEBPA mutations were included in the analysis. In CEBPA wildtype patients, ID1 expression had no impact on complete remission‐rate, OS or RFS. In conclusion, CEBPA mutations seem to deregulate ID1 expression. Therefore, ID1 expression is not an independent prognostic factor in CN‐AML.

The hypomorphic TERT A1062T variant is associated with increased treatment-related toxicity in acute myeloid leukemia

Hypomorphic germline variants in TERT, the gene encoding the reverse transcriptase component of the human telomerase complex, occur with a frequency of 3–5% in acute myeloid leukemia. We analyzed the clinical and prognostic impact of the most common TERT A1062T variant in younger patients with acute myeloid leukemia intensively treated within two prospective multicenter trials. Four hundred and twenty patients (age 17–60xa0years) were analyzed for the TERT A1062T variant by direct sequencing. Fifteen patients (3.6%) carried the TERT A1062T variant. Patients with the TERT A1062T variant had a trend towards less favorable and more intermediate 2/adverse karyotypes/genotypes according to the European Leukemia Net classification. In univariate and multivariate analysis, patients with the TERT A1062T variant had a significantly inferior overall survival compared to wild-type patients (6-year overall survival 20 vs. 41%, pxa0=xa00.005). Patients with the TERT A1062T variant showed a high rate of treatment-related mortality: 5/15 (33%) died during induction therapy or in complete remission as compared to 62/405 (15%) of the wild-type patients. In patients with the TERT variant, 14/15 (93%) suffered from non-hematological/non-infectious grade 3/4 adverse events (mostly hepatic and/or mucosal) as compared to 216/405 (53%) wild-type patients (pxa0=xa00.006). In multivariate analysis, the TERT A1062T variant was an independent risk factor predicting for adverse events during induction chemotherapy. In conclusion, the TERT A1062T variant is an independent negative prognostic factor in younger patients with acute myeloid leukemia and seems to predispose those patients to treatment-related toxicity.