ALBERT

Beschreibung: Key Points IFN-γ impairs maintenance of HSCs by directly reducing their proliferative capacity and impairing their restoration upon viral infection. IFN-γ induces SOCS1 expression in HSCs, which inhibits TPO-induced STAT5 phosphorylation, thereby deregulating key cell-cycle genes.

Beschreibung: Abstract 3521 Background Acute promyelocytic leukemia (APL) accounts for approximately 5% of all acute myeloid leukemias (AML). The characteristic molecular feature of APL is a fusion product named PML-RARA which acts as transcriptional repressor that affects gene expression patterns involved in differentiation, apoptosis, and self-renewal. The internal tandem duplication of the Fms-related Tyrosine-like Kinase 3 (FLT3-ITD) confers a poor prognosis in non-APL AML, however its effect in APL is still under discussion as several investigators found no prognostic influence for FLT3-ITD in APL. Aberrant DNA-promotor-methylation of tumor suppressor genes contributes significantly to leukemogenesis and oncogenic transformation. Deneberg et al. recently identified characteristic methylation profiles for cytogenetically normal AML, however no specific methylation profile was associated with FLT3-ITD in a study that excluded APL. To further elucidate the influence of aberrant methylation in FLT3-positive APL we carried out a genome wide DNA methylation analysis on APL samples with and without FLT3-ITD. Methods In total, genomic DNA from blasts of 54 APL patients at initial diagnosis (bone marrow n=32, peripheral blood n=22) were analyzed (median age 46 years, gender: 35 female, 19 male, blast count median 80%). The molecular analysis was carried out with written informed consent, with permission of the institutional review board and in accordance with the declaration of Helsinki. DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 25% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Pyrosequencing was performed to validate methylation changes as detected by the array-based analysis. Results The methylation pattern of FLT3-ITD-positive APL (n=18) patients was analyzed and compared to patients without FLT3-ITD (n=32) or D835 Mutation (n=4). We identified 133 CpGs that were significantly differentially methylated in FLT3-ITD-positive APL as compared to FLT3-ITD-negative APL. The most significant differential methylation was observed for 5 CpGs showing a strong hypomethylation of the chemokine (C-C motif) receptor 6 (CCR6) in FLT3-ITD-APL as compared to FLT3-negative APL (q-value 〈 6.9 *10−13). Other interesting target genes showing pronounced hypomethylation in FLT3-ITD positive APL samples belonged to the family of phosphatases such as the dual specificity phosphatase 5 (DUSP5), protein tyrosine phosphatase, receptor type, N polypeptide 2 (PTPRN2) and protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). The most prominent hypermethylation in FLT3-ITD APL was observed in CpGs within the coding region of suppressor of cytokine signaling 2 (SOCS2) and significantly discriminated between FLT3-ITD and FLT3-negative APL (q-value 〈 10−5). The results of the genome-wide analysis obtained with the Illumina 450K BeadChip were validated for 4 CPGs in 10 samples via pyrosequencing and showed a robust Pearson correlation of 0.92 suggesting a good and reliable performance of the Illumina 450 K Bead Chip Assay. Conclusions The current study represents a comprehensive genome wide methylation analysis of a clinically well-defined cohort of APL patients. We here demonstrate for the first time that in contrast to cytogenetically normal AML, APL patients with FLT3-ITDs display a highly specific and disease defining DNA methylation profile. Thereby key regulators of cellular growth signaling such as SOC2, PTPRN2 and DUSP5 are significantly differentially methylated in dependency of FLT3-ITD status. This suggests that a cooperative effect between PML-RARA and FLT3-ITD is mediated by dysregulation of DNA methylation. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 4235 Red blood cell production is a strictly regulated process and homeostatic maintenance of the erythropoietic system requires equilibrium between the rate of erythroid cell production and red blood cell destruction. Hematopoietic cytokines play a crucial role in regulating expansion, differentiation and survival of erythrocyte progenitors. Shortage of growth factors triggers the mitochondrial apoptosis pathway, which is critically dependent on Bcl-2 family members. However, the contribution of this mechanism in the regulation of erythropoiesis remains ill-defined. This prompted us to screen for candidate genes involved in this process in erythroid progenitors. We found that the expression of Noxa, a pro-apoptotic Bcl-2 family member, is upregulated during erythroid differentiation and following cytokine-withdrawal in erythroid progenitor cells. Knockdown or deletion of Noxa in IL-3 dependent human and murine erythroid progenitor cell lines increased Mcl-1 levels, which correlated with markedly decreased apoptosis following cytokine withdrawal. Importantly, Noxa ablation in mice increased extra-medullary erythropoiesis, resulting in enhanced numbers of early splenic erythroblasts and circulating reticulocytes. Noxa-deficient hematopoietic progenitors were more resistant to apoptosis induced by growth factor deprivation and displayed increased colony-forming potential. In addition, combined loss of Noxa and Bim resulted in enhanced resistance of erythroid progenitors to cytokine withdrawal compared to WT or single Bim knockouts, suggesting a non-redundant role for Noxa and Bim in regulating survival of erythroid progenitors in response to cytokine deprivation. Finally, in a model of acute haemolytic anaemia, deletion of Noxa enhanced subsequent hematocrit recovery. Together, these findings identify a non-redundant role for BH3-only protein Noxa in the regulation of erythroblast survival during early erythropoiesis. Therefore, Noxa may be a novel component to control red blood cell numbers and modulation of this pathway could be envisaged in therapeutic options for treatment of anaemia. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1295 Poster Board I-317 Introduction Recent animal studies suggest that measurable amounts of factor VIIa and antithrombin (AT) complexes are formed and accumulate following rFVIIa administration. The in vivo rate of inhibition has been reported to be faster than the un-stimulated in vitro reaction between AT and free rFVIIa and of the same order of magnitude as the rate determined in the presence of tissue factor. To study the impact of AT inhibition on the elimination of rFVIIa in humans, we measured the pharmacokinetics (PK) of rFVIIa and rFVIIa-AT complex formation in 10 hemophilia A or B patients. Patients and Methods The PK of single-dose rFVIIa 90 μg/kg (Novo Nordisk A/S) was evaluated in 10 severe FVIII- or FIX-deficient patients in a non-bleeding state. The plasma concentrations of FVIIa activity (FVII:C), FVII antigen (FVII:Ag), FVIIa-AT, D-dimer and F1+2 fragment were determined immediately before, and at 0.5, 1, 2, 4 and 6 hours following rFVIIa dosing. Results Significant amounts of FVIIa–AT complex were formed in vivo after rFVIIa administration, and reached a maximum of 5.4 ± 0.8 nmol/L [mean ±SD] at 2 hours following rFVIIa administration and declined to 4.4 ± 0.9 nmol/L at 6 hours, as compared to 0.1 ± 0.05 nmol/L at baseline. While the FVII:C PK data in this study were consistent with previous data, there was greater total body clearance (Cltot), a larger volume of distribution (Vdss) and a shorter plasma half-life (T1/2) of FVII:C relative to FVII:Ag (Table). No change in D-dimer was observed after the administration of rFVIIa, while a slight increase in F1+2 fragment levels to 258 ± 73 pmol/L was measured 4 hours after rFVIIa dosing, as compared to 141 ± 45 pmol/L at baseline. Conclusion A significant divergence between the clearance of rFVIIa, as determined by either FVII:C or FVII:Ag measurements, can be accounted for by AT complex formation. Inhibition by AT appears thus to have a significant impact on the elimination of FVII:C activity from the circulation when rFVIIa is administered at a therapeutic dose. Similar to animal data, the formation of the FVIIa-AT complexes in vivo was faster than anticipated from in vitro studies, indicating that the exposure to the vessel wall stimulates the FVIIa inhibition by AT. Analyses of coagulation parameters did not indicate induction of systemic coagulation. Disclosures Ezban: NovoNordisk A/S: Employment. Pelzer:NovoNordisk: Employment. Agerso:NovoNordisk: Employment. Petersen:NovoNordisk: Employment. Hedner:NovoNordisk: Employment. Carr:NovoNordisk: Employment.

Beschreibung: To explore whether and how T cells can affect myelopoiesis, we investigated myeloid differentiation in a model for T cell-mediated immune activation. We found that CD70-transgenic (CD70TG) mice, which have elevated numbers of interferon-γ (IFN-γ)–producing effector T cells in the periphery and bone marrow, are almost devoid of eosinophilic granulocytes. Induction of allergic airway inflammation in these mice failed to induce eosinophilia as well as airway hyperresponsiveness. CD70TG mice also have strongly reduced numbers of eosinophil lineage-committed progenitors, whereas granulocyte/macrophage progenitors from these mice are unable to generate eosinophils in vitro. We found that granulocyte/macrophage progenitors express IFN-γR1 and that IFN-γ is sufficient to inhibit eosinophil differentiation of both murine and human progenitor cells in vitro. We demonstrate that inhibition of eosinophil development in CD70TG mice is IFN-γ–dependent and that T cell–derived IFN-γ is sufficient to inhibit eosinophil formation in vivo. Finally, we found that IFN-γ produced on anti-CD40 treatment and during viral infection can also suppress eosinophil formation in wild-type mice. These data demonstrate that IFN-γ inhibits the differentiation of myeloid progenitors to eosinophils, indicating that the adaptive immune system plays an important role in orchestrating the formation of the appropriate type of myeloid cells during immune activation.

Beschreibung: Introduction Total (-7) or partial (7q-) monosomy 7 is frequent in malignant myeloid disorders, observed in around 12% of MDS/AML and up to 40% of therapy-associated MDS/AML. Monosomy 7 is associated with poor outcome, high susceptibility to infections and poor response to chemotherapy. A therapeutic benefit for 5-azacytidine was previously described (Fenaux et al., 2009). The present study was designed to analyze clinical features, prognosis and response to different therapeutic strategies in patients with monosomy 7 in a multicentric, retrospective German cohort study. Patients and methods Currently, 231 patients with MDS/AML following MDS and monosomy 7 were included. Inclusion criteria were defined as follows: Morphologic diagnosis of MDS/AML following MDS, age ≥18 years, bone marrow blast count ≤30% and presence of -7 or 7q-. The data was assembled from centers in Düsseldorf, (n=120; 52%), Cologne (n=38; 17%), Freiburg (n=31; 13%), Göttingen (n=14; 6%), Munich (n=13; 6%), Dresden (n=11; 5%) and Mannheim (n=4; 2%). The median age in the study cohort was 67 years, 65% of patients were males. 29/231 patients (13%) were diagnosed as AML following MDS. MDS/AML was therapy-associated in 24 patients (11%). Regarding IPSS, 38 (19%) were classified as low/intermediate 1 risk and 165 (81%) as intermediate-2/high-risk. According to IPSS-R, 2 (1%) were assigned to the very-low/low risk group, 31 (16%) to the intermediate group, 52 (27%) to the high-risk group and 107 (56%) to the very high risk group. The treatment was classified as follows: Best supportive care (BSC), low-dose Chemotherapy (LDC), high-dose chemotherapy (HDC), demethylating agents (DMA; either 5-azacytidine or decitabine), and others. Results A best supportive care regimen was chosen in nearly half of the patients (49%). The remaining patients received 1-4 sequential therapies (1: 29%; 2: 11%; 3: 10%; 4: 1%). As the first line therapy, 64 patients (54%) received DMA, 24 (20%) an allo-Tx, 9 (8%) HDC, 5 (4%) LDC, and 16 (14%) were treated with other therapies. The best prognosis was observed in patients eligible for allo-Tx: The median OS in transplanted patients was 924 days as compared to 361 days (p

Beschreibung: Multidrug-resistant bacterial pathogens (MRP) such as extended-spectrum beta-lactamase producing enterobacteriaceae (ESBL), vancomycin-resistant enterococci (VRE) and methicillin-resistant staphylococcus aureus (MRSA) are an emerging challenge in allogeneic hematopoietic cell transplantation (HCT). However, to our knowledge there are no data in the existing literature on the prevalence of MRP and of the impact of these multidrug-resistant pathogens on the outcome after allogeneic HCT. Thus, it was the purpose of this study to systematically analyze the issue of MRP in patients who underwent allogeneic HCT. PATIENTS AND METHODS: From 06/2010 to 12/2013 a total number of 72 (F: n=23; M: n=49) consecutive patients who received the first allogeneic HCT at our institution were retrospectively analyzed. The underlying diseases were AML (n=44), ALL (n=5), CML (n=4), MPN (n=2), lymphoma (n=5), MDS (n=9), and multiple myeloma (n=3). The conditioning regimen was myeloablative in 23 patients and reduced intensity in 49 patients. Patients were transplanted with peripheral blood stem cells (n=69) or bone marrow (n=3) from matched siblings (n=19), matched unrelated (n=45), mismatched (n=5) or haploidentical donors (n=3). As baseline investigation before commencing with the conditioning all patients underwent a comprehensive screening for MRP, i.e. ESBL, VRE and MRSA. For that reason swabs from nose, throat, axilla, urethra and anus as well as stool and urine were collected. The same screening was performed at discharge from hospital after allogeneic HCT and in case of a new admission into our institution. In addition routine microbiological investigations such as bacterial cultures from blood, urine, swabs, stool or central venous catheters were performed whenever clinically needed. Multidrug-resistant gram negative bacteria were categorized as 4MRGN (resistant to cephalosporins, piperacillin, fluorochinolones and to carbapenems) or as 3MRGN (resistant to 3 of these 4 antimicrobial drug groups). The primary endpoint of this analysis was day 100 non relapse mortality (NRM). Secondary endpoints were NRM and overall survival (OAS) two years post HCT. RESULTS: 23 out of 72 patients (32%) were colonized by multidrug-resistant bacterial pathogens (MRP+ group) either at baseline (baseline MRP+ group, n=13, 18%) or at any other time point until day 100 post transplantation. Four patients were positive for two MRP either simultaneously at baseline (n=1) or at different time points (n=3). Detected MRP (n=27) were as follows: 3MRGN Escherichia coli or Klebsiella pneumonia (n=11), 4MRGN Pseudomonas aeruginosa (P. aeruginosa, n=4), 3MRGN P. aeruginosa (n=2), 4MRGN Stenotrophomonas maltophilia (n=1), 3MRGN Citrobacter freundii (n=1), VRE (n=7) and MRSA (n=1). Out of these 23 patients 7 patients developed an infection with MRP after HCT: Septicemia with 3MRGN Escherichia coli (n=3), septicemia with 3MRGN Klebsiella pneumonia (n=1), septicemia with P. aeruginosa (4 MRGN n=2, 3MRGN n=1) and one patient with VRE septicemia and 4MRGN P. aeruginosa pneumonia. Out of the 4 patients with multidrug-resistant P. aeruginosa infection three died transplant related (two of these patients had been already colonized with 4MRGN P. aeruginosa at baseline). However, 2-year OAS of MRP colonized versus non-colonized patients was essentially the same (66.6% versus 63.0%, median follow up 23.8 months range 7.0 to 48.0 months). Day 100 NRM was higher in the baseline MRP+ group and in the entire MRP+ group in comparison with non-multidrug-resistant bacterial pathogens colonized patients (23.1% and 17.4% versus 10.2%, not statistically significant [ns]). Data for 2 year NRM were 32.7%, 22.2% and 17.1% (ns), respectively. The increased NRM of MRP+ patients was mainly due to the high NRM of patients infected by multidrug resistant P. aeruginosa. CONCLUSIONS: Colonization or infection with 3MRGN gram negative non-P. aeruginosa enterobacteriaceae or by VRE has no negative impact on the outcome after allogeneic HCT. Thus allogeneic HCT of patients colonized by MRP is feasible. However, patients colonized by multidrug-resistant P. aeruginosa seem to have a dismal outcome. Allogeneic HCT of these patients should be considered with care. We therefore suggest to include screening for MRP in the pretransplant recipient work up particularly to identify patients colonized by multidrug-resistant P. aeruginosa. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Previous studies showed that the HLA class I region is associated with Epstein-Barr virus (EBV)–positive Hodgkin lymphoma (HL) and that HLA-A is the most likely candidate gene in this region. This suggests that antigenic presentation of EBV-derived peptides in the context of HLA-A is involved in the pathogenesis of EBV+ HL by precluding efficient immune responses. We genotyped exons 2 and 3, encoding the peptide-binding groove of HLA-A, for 32 single nucleotide polymorphisms in 70 patients with EBV+ HL, 31 patients with EBV− HL, and 59 control participants. HLA-A*01 was significantly overrepresented and HLA-A*02 was significantly underrepresented in patients with EBV+ HL versus controls and patients with EBV− HL. In addition, HLA-A*02 status was determined by immunohistochemistry or HLA-A*02–specific polymerase chain reaction (PCR) on 152 patients with EBV+ HL and 322 patients with EBV− HL. The percentage of HLA-A*02+ patients in the EBV+ HL group (35.5%) was significantly lower than in 6107 general control participants (53.0%) and the EBV− HL group (50.9%). Our results indicate that individuals carrying the HLA-A*02 allele have a reduced risk of developing EBV+ HL, while individuals carrying the HLA-A*01 allele have an increased risk. It is known that HLA-A*02 can present EBV-derived peptides and can evoke an effective immune response, which may explain the protective phenotype.

Beschreibung: Introduction With current therapy regimens over 75% of patients with de novo acute promyelocytic leukemia (APL) can be cured. Approaches to further improve patient outcome by stratifying patients at the time of initial diagnosis according to their individual risk and to adjust therapy accordingly have been based on clinical features only. Molecular markers have not been established for risk stratification as yet. Recently, we have shown that high expression levels of the genes brain and acute leukemia, cytoplasmic (BAALC) and ets related gene (ERG) are associated with inferior outcome in APL patients. In addition, data indicate that aberrant expression of the gene Wilms’ tumor 1 (WT1) is a negative prognostic factor with regard to overall survival (OS) after complete remission (CR) and relapse free survival (RFS) in APL. In this study we evaluated the prognostic relevance of a combined score integrating the expression levels of the above mentioned genes to further improve risk stratification in APL patients. Methods Expression levels of BAALC, ERG and WT1 of 62 patients with newly diagnosed APL were retrospectively analyzed in bone marrow mononuclear cells using multiplex reverse transcriptase quantitative real-time PCR (qRT-PCR). Median age of patients was 47 years (range: 19 to 82y). All patients gave informed consent. Patients were diagnosed and treated in the German AML Cooperative Group (AMLCG) study with a treatment of simultaneous ATRA and double induction chemotherapy including high-dose ara-C, consolidation and maintenance chemotherapy. The following gene expression levels were identified as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression 〉75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low/high). A risk score was developed as follows: for the presence of one of the mentioned risk factors one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low/high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors) could be allocated to one patient. Accordingly, patients were divided into four risk groups: 7 patients scored 0 points (= low risk), 27 patients scored 1 point (= intermediate 1 risk), 19 patients scored 2 points (= intermediate 2 risk) and 9 patients scored 3 points (= high risk). Subsequently, OS, RFS and relapse free interval (RFI) were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the four risk groups (p