ALBERT

Description: Key Points Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases. Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.

Description: 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.

Description: Myelodysplastic syndromes (MDS) are mainly characterized by dyserythropoiesis resulting in anemia. So far, this pathological hallmark is incompletely understood. Notch signalling has recently been linked to impaired erythropoiesis and megakaryopoiesis of CD34+ progenitor cells, however its role in MDS is unclear. On the other hand, in MDS demethylating therapy results in decreased transfusion dependency, indicating aberrant methylation of key erythroid genes. Therefore, we have analyzed Notch pathway elements and its association with the key erythroid factors GATA1 and BCLxl and examined the methylation of CpGs flanking cis-regulatory elements (including N-box suppressor binding site for HES1 and GATA box) of the GATA1 erythroid promoter in differentiating CD34+ cells selected from MDS patients. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO. Cell harvest was at days 0, 4, 7 and 11. RNA-expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). GPI was used as a housekeeping gene. DNA methylation at 7 CpGs of the GATA1 gene promoter was quantitatively analyzed by Pyrosequencing (Pyro Mark ID, Biotage, Uppsala, Sweden) of bisulfite treated genomic DNA at any specific time point. In normal erythropoietic cells, RNA expression of GATA1 and of BCLxl was steadily up regulated, particularly during late erythropoietic differentiation. In contrast, during MDS erythropoiesis a loss of typical up regulation of GATA1 (day 11: 2.08 vs. 0.11; p=0.001) and BCLxl (day 11: 7.46 vs. 0.16; p=0.0005) was observed. Notch ligand DLK1 showed increased expression during erythropoiesis particularly in high risk MDS as compared to normal controls (days 4–11: 0.38 vs. 0.03; p=0.02). Furthermore, expression of HES1 was increasing during the course of normal erythropoietic differentiation but not in lineage specific cells from MDS patients (day 11: 0.01 vs. 0.006; p=0.1). No hypomethylation of CpGs flanking repressor HES1 binding site within the 5′-GATA region was detected in MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up regulation in contrast to MDS erythropoiesis (day 11: 26% vs. 58%; p=0.00004). Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fail to be up regulated during MDS erythropoiesis. The higher residual 5′-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggests a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS. However, a transcriptional activation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 and a hypomethylation of its binding site could not be detected in MDS.

Description: Introduction: Disturbed proliferation and differentiation are the most crucial oncogeneic factors leading to malignant turnover of hematopoiesis in myeloid malignancies. Therefore, estimating the lifetime proliferation status of malignant hematopoietic cells is critical. Recently the hypothesis of an epigenetic molecular clock has been corroborated. Depending on the accumulation of CpG methylation errors throughout life after each cell division it is possible to measure an increased DNA methylation of formerly unmethylated CpG islands and subsequently relate it to the mitotic cell age. In order to elucidate the importance of disturbed proliferation in hematologic diseases we initiated a novel approach for profiling mitotic ages of hematopoietic cells in myelodysplastic syndrome and acute leukemia. Patients & Methods: Bone marrow (BM) cells of patients with myelodysplastic syndrome (MDS, IPSS-low/int-1-risk n=23, IPSS-int-2/high-risk n=27), acute myeloid leukemia (AML, n=55), acute lymphoblastic leukemia (ALL, T-lineage n=40, B-lineage n=8), and of age matched healthy individuals (n=24) were analyzed. In addition, selection of CD34+ cells was performed in MDS (n=43), in AML (n=10) as well as in healthy BM samples (n=31). CD19+ peripheral blood cells from healthy donors (n=13) served as an additional control. Genomic DNA was isolated and bisulfite converted using standard TRIZOL technique (Invitrogen, Carlsbad/CA, USA) followed by EpiTect-Bisulfite-Kit conversion (Qiagen, Hilden, Germany). PCR amplification of a CpG rich 3′ site of the Cardiac Specific Homeobox gene (CSX), considered as an epigenetic molecular clock locus, was performed as previously reported. DNA methylation was quantitative measured using the PyroMark ID Pyrosequencing system (Biotage, Uppsala, Sweden). Quantitative DNA methylation data are presented with mean ± S.E.M. Results: In MDS int-2/high-risk specific DNA methylation of BM (26.6 ± 1.8 %) and CD34+ (28.6 ± 2.7 %) was significant higher compared to low/int-1-risk MDS (BM: 19.2 ± 1.6 %, p=0.0047, CD34+: 18.7 ± 2.4 %, p=0.0093) and healthy donors (BM: 17.8 ± 0.5 %, CD34+: 17.0 ± 0.4 %, p

Description: Introduction Recently, Erythroferrone (ERFE) was discovered as a new regulator of hepcidin in the context of hematopoietic stress and erythropoietin (EPO) stimulation (Kautz et al., Nature Genetics 2014). ERFEhas been shown to be expressed by erythroprogenitor cells of the bone marrow in response to increased erythroid activity induced by phlebotomy, EPO treatment or simulation of infectious situations in mice. It induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. Myelodysplastic Syndromes (MDS) are a group of heterogeneous malignant hematologic diseases characterized by inefficient hematopoiesis, severe anemia and deregulated iron homeostasis. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of ERFE in different hematopoietic compartments of MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival. Methods CD71+ erythroprogenitor cells (n=198 samples) were immunomagnetically purified from mononuclear bone marrow (BM) cells of a total of n=148 MDS and n=18 sAML patients. Chronological samples were available in n=21 cases. For controls, CD71+ BM cells were analyzed from n=35 healthy donors. In addition to CD71+ cells, CD61+, CD15+ , CD34+, selected from BM, as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old volunteers. After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR. Results In comparative expression analyses of different hematopoietic BM progenitor fractions (CD34+, CD15+, CD61+ and CD71+), ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression profiles in the CD71+ subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.3, p

Description: Introduction Deletion of chromosome 5q (del(5q)) defines a distinct clinical subtype of myelodysplastic syndromes (MDS) and qualifies patients to specific treatment with Lenalidomide (LEN). Therefore, detection and monitoring of this deletion is an important element in routine clinical diagnostics for determining molecular response. Current methodologies for performing these analyses consist of cytogenetics, fluorescence in situ hybridization (FISH) or microarrays. All of these methods have downsides due to the high demands to the input material, i.e. viable cells or necessity for large amounts of high quality genomic DNA (gDNA). To perform quantitative assessment of cytogenetic lesions in low quantity or residual material we here present the establishment of a PCR-based assay for interrogation of del(5q) in MDS, based on the allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Methods Genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of n=86 MDS del(5q) patients. 49 non-del(5q) MDS patients were used as controls. Serial chronological BM samples (n=95) following treatment with LEN from n=40 del(5q) patients, who were enrolled in the LEMON-5 trial from the German MDS study group, were analysed. Using 10ng DNA, 12 fluorochrome-labelled PCR amplicons of STR loci located between chromosomal bands 5q21 and 5q31 were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis and allele size quantification of heterozygous STR loci was performed. Finally, the degree of skewing in the allelic ratios of all informative STR markers was averaged and translated into an allelic burden of del(5q). Results Paired quantitative correlation of clone sizes using STR-PCR and interphase FISH was carried out in n=34 samples and revealed highly concordant results with r²=0.924. The diagnostic accuracy of the PCR assay was evaluated by receiver operating characteristic (ROC) analysis and revealed an area under the curve of 0.989 (sensitivity and specificity of 0.977 and 0.948, respectively). Prior to treatment with LEN, clone sizes as determined by STR-PCR were heterogeneous (mean: 57%, range: 11-91%). During follow-up analysis, while cytogenetic analyses failed (e.g. metaphase failure) in 7/40 (18%) cases, our STR-PCR assay successfully generated estimates of del(5q) cell burden in all available samples. Upon LEN treatment, n=12 patients achieved major cytogenetic remission (absence of del(5q)-positive metaphases). The mean clone size carrying del(5q) determined by STR-PCR in that group was 7% (range 3 - 10%) and significantly increased compared with n=15 patients who reached minor cytogenetic response (defined as 50% reduced aberrant metaphases, mean 13%, range 5 - 39%, p=0.025). Intriguingly none of n=6 patients without cytogenetic response achieved a del(5q) clone size of less than 35% as determined by STR-PCR (mean 46%, range 35 - 66%), highlighting the correlation of PCR based follow-up analysis with currently used cytogenetic methods for response evaluation. Finally in n=93 matched PB and BM samples a correlation of del(5q)-frequency in BM versus PB showed r²=0.81. Moreover, in 96% of samples in which the BM still showed clone sizes 〉10%, we reliably detected del(5q) in corresponding PB cells with a robust sensitivity of 5% deleted cells. Discussion We present a highly adaptable tool for precise measurement of large chromosomal deletions, requiring only minute amounts of genomic DNA. It shows a very good quantitative correlation with established methods and good diagnostic accuracy. Most importantly, this PCR based assay does not require dividing cells so it can be performed from PB, which shows a sufficient correlation with clone sizes in BM and rarely involves the risk of underrepresentation of del(5q)-clones in PB, possibly allowing the use of PB as a regular specimen for clone size monitoring. Thus, especially in the context of serially monitored patients this assay represents an alternative method for less invasive tracking of cytogenetically aberrant clones. Disclosures Platzbecker: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Schlenk:Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Introduction: Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm. Methods: We collected BM samples from voluntary healthy young adults (age = 24 - 25 years, female n=3, male n=3), healthy old adults (age 66 - 79 years, female n=3, male n=3) and patients with very low - intermediate risk MDS (age 51 - 87 years, female n=3, male n=3). After isolation of BM mononuclear cells by Ficoll gradient centrifugation, 5x106 mononuclear BM cells were seeded into 25cm² flasks and cultured using StemMACS human MSC Expansion Media (Miltenyi Biotec) with weekly media exchange to select for MSCs. These were expanded and harvested in passage 2. Absence of residual hematopoietic cells was controlled by FACS with anti CD45, CD31, and CD146. Whole transcriptome RNA-sequencing on all samples was carried out from 150ng of high quality RNA using the TruSeq stranded total RNA protocol and 100bp paired end sequencing (Illumina). The bio-informatical pipeline consisted of mapping using hisat2 and cufflinks for calculation of differentially expressed genes. Results: RNA-sequencing generated a mean of 94 million reads per sample. Between the groups "healthy young" and "healthy old" 331 differentially regulated genes were identified. Between "healthy old" and "MDS" 514 genes were differentially regulated (fold change 〉 1.5, false discovery rate, FDR 〈 0.05). Among these, 197 genes were differently expressed between all three groups. With these parameters, a total of 17 genes showed a continuous and significant increase of expression from healthy young over healthy old toward MDS. Among these were Kit ligand (KITLG) but also a cluster of membrane based cell adhesion molecules such as Cadherin-6 (CDH6), Laminin Subunit Alpha 2 (LAMA2) and Laminin Subunit Gamma 2 (LAMC2) and others. Conversely, 5 genes showed a continuous and significant decrease of expression from healthy young over healthy old toward MDS, among these Leukocyte-specific protein 1 (LSP1), a gene implicated in regulation of T-cell migration. Gene set enrichment analysis revealed that MDS MSCs exhibited a significant depletion of genes involved in early adipogenic differentiation and enrichment of gene sets associated with extracellular matrix remodeling (FDR 〈 0.05, normalized enrichment score 〉 1.7). Although cells were cultured under normoxic conditions, MDS-MSCs displayed marked intrinsic feature of hypoxia. Conclusion: By integrating transcriptomic data from BM stroma cells from healthy individuals during aging and comparison to BM stroma cells from MDS patients we have identified gene sets that are significantly differentially expressed per continuitatem. On the background of the hypothesis that molecular changes in the microenvironment of MDS are an exacerbation of changes also taking place during normal aging in the bone marrow, these genes, which are accumulated in the context of extracellular matrix and cell adhesion are promising candidates to further elucidate a BM stroma based pathogenesis of MDS. Disclosures No relevant conflicts of interest to declare.

Description: Identification of common genomic lesions in progenitor cells of MDS Patients could lead to the discovery of new target genes in this disease and may be of prognostic value. Therefore, we carried out a detailed genome-wide mapping of genomic DNA from highly purified CD34+ progenitor cells from MDS patients and healthy individuals with high-resolution single nucleotide polymorphism (SNP) microarrays which scan 500,000 SNPs with a median inter-SNP distance of approximately 2.5 kb. Bone marrow aspirates were obtained from 14 MDS patients (IPSS low risk n=6, high risk n=8) and 6 healthy individuals after informed consent. CD34+ cells were purified by high gradient magnetic cell separation. Genomic DNA and RNA were extracted with standard TRIZOL technique and quality controlled with the Agilent Bioanalyzer 2100 and Nanodrop ND-1000 systems. 500 ng of each of the genomic DNA were processed according to the protocol of the Affymetrix 500 k NspI and StyI genomic mapping protocol, hybridized to 500 k NspI/StyI chip sets and scanned on an Affymetrix GeneChip scanner 3000. The median SNP call rate of analysed samples was 88.6% and ranged from 76.3% to 95.4%. One sample from the MDS patients and two samples from the healthy donors were excluded from analysis due to insufficient call rates. Raw signal intensity data was generated by the GCOS 4.0 software and imported into Partek Genomics 6.2 software. The control samples of healthy individuals were assigned a copy number of two and used as a reference baseline to calculate copy numbers in MDS samples. On the calculated values genomic smoothing was performed with a window width of 0.5 Mbps and a Gaussian width at half maximum 50% of window width. Significant regions of copy number alterations were calculated with a test region width of 0.5 Mbp and contiguous regions set to contain at least 1 Mbp (p

Description: Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid malignancies characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. Particularly early stage MDS are at least in part characterized by an increased apoptosis of myeloid and erythroid progenitors that causes peripheral cytopenia. APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (Fas receptor) and the Fc domain of human IgG1. APG101 effectively binds to the CD95 ligand (CD95L) expressed on effector cells as well as to functionally active ligand in solution, by that blocking the interaction between CD95 and its ligand. The aim of our study was to evaluate whether APG101 treatment of primary CD34+ reduces the apoptotic rate and improves the differentiation capacity of these cells. Methods Bone marrow cells were obtained during routine bone marrow aspiration after all patients gave their written informed consent. Isolated primary CD34+ cells from 11 MDS patients were cultured in complete supplemented IMDM medium for 6 days with increasing concentrations of APG101 (1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL). After incubation time, cells were multicolor- stained with the following dye combination: Annexin-FITC + CD235a-PE + CD34-PECy7 + CD71-APC + 7-AAD and analyzed immediately on a flow cytometer (FACSCanto, BD Bioscience, Heidelberg, Germany). Analysis of raw FACS data was done with the FACSDiva software. To analyze the differentiation capacity of CD34+ progenitors, methylcellulose assays were performed in parallel to the aforementioned experiments. However, due to limited cell numbers, colony assays were performed on 9 MDS patients only. Cells were cultured in triplicates with increasing concentrations of APG101 for 14 days. Colonies were counted and the mean number of colonies was determined. Results Treatment of differentiating CD34+ cells with APG101 led to a decreased apoptosis in both CD34+ cells and CD71+ cells, respectively, indicated by decreased Annexin-FITC fluorescence. Interestingly, this effect was particularly seen at low APG101 concentrations (maximum of 10 µg/ml), while the effect was abrogated at higher APG101 concentrations. The anti-apoptotic effect was more pronounced in low risk MDS patients compared to high risk MDS patients. No effect was seen when the CD235a+ fraction of cells was analyzed. With regard to colony formation, an improvement of erythroid differentiation, indicated by an increase in CFU-E, was found in 3 out of 4 low risk patients (less than 5% blasts in the bone marrow). No effect was seen on erythroid differentiation in high risk patients (more than 5% blasts in the bone marrow). Conclusion APG101 shows promising in vitro activity in viable CD34+ cells with regard to inhibition of apoptosis and promotion of differentiation. The observation that the anti-apoptotic effect was more pronounced in low risk MDS patients as compared to high risk MDS patients supports the concept of increased apoptosis particularly in early stage MDS progenitors. Although the numbers in the differentiation experiments are small, we found a promising effect of APG101 on CFU-E formation at lower doses in patients with less than 5% bone marrow blasts. Moreover, the same dose-dependent effect was observed in the apoptosis assays. Since the activation of the CD95 pathway seems not only to be involved in apoptosis induction, but is also required for terminal erythroid differentiation in normal hematopoiesis, this dose-dependent effect might particularly reflect these ambivalent roles of CD95 and its ligand in both MDS and healthy hematopoiesis, repsectively. Disclosures: Kunz: APOGENIX GmbH: Employment. Fricke:APOGENIX GmbH: Employment.