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: 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

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: Risk stratification in acute promyelocytic leukemia (APL) is based on clinical parameters, namely leukocyte and platelet counts at initial diagnosis as combined in the Sanz Score. However, during the last years the influence of additional molecular genetic markers on prognosis of APL patients has been postulated. In 2015 we published the results of a molecular risk score integrating expression data of the genes brain and acute leukemia, cytoplasmic (BAALC), ets' related gene (ERG) and Wilms' Tumor 1 (WT1) with strong independent influence on outcome and relapse risk of APL patients treated in the German AMLCG studies (Hecht et al., Leuk Res 2015). The aim of our study was to validate our data in an independent patient cohort. Methods: In cooperation with the German SAL (Study Alliance Leukemia) group we obtained a validation set of samples of mononuclear cells derived from the bone marrow of 76 patients with confirmed diagnosis of APL prior to therapy. The validation cohort consisted of 37 female and 39 male patients with a median age of 50 years (range 20-82 years; Table 1). Patients were diagnosed and treated between 2000 and 2014 mostly with an ATRA plus Idarubicin based induction therapy followed by Sanz score-dependent consolidation and maintenance chemotherapy. RNA was extracted using the AllPrep DNA/RNA Mini Kit and cDNA was synthesized using 500µg of RNA with a QuantiTect Reverse Transcription Kit (both Qiagen, Hilden, Germany). Expression levels of BAALC, ERG and WT1 were then analyzed using quantitative real-time RT-PCR with the same conditions and primers as used in the original test cohort and the same calibrator cDNA from cell lines was used for the analysis. The following gene expression levels were defined 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 or high). The integrative risk score was calculated as described before: 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 or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors). Overall survival (OS), relapse free survival (RFS) and cumulative incidence of relapse (CIR) were calculated using the Kaplan-Meier method. Results: The expression of the three genes BAALC, ERG and WT1 compared to healthy controls were exactly the same as in the original cohort. Application of the molecular risk score on APL patients of the validation cohort clearly discriminated patients according to their risk comparable to the original APL cohort (Figure 1). Patients with 0 points had a very good prognosis with no deaths or relapses, whereas patients with 3 points (i.e. concurrent presence of all three risk factors) had a very poor outcome with an OS of 51%, a RFS of 50% and a CIR of 38%. The differences between patients with 1 and 2 points were less pronounced in the validation cohort. However, the statistical analyses in the validation cohort did not yield significance. As the main reason we assume that the validation cohort altogether showed a significantly improved OS and RFS compared to the original cohort which was treated a decade earlier, so differences between the subgroups could not be that pronounced. For both cohorts stratification by the Sanz Score did not show significant differences in outcome (analysis for RFS p=0.46 in original cohort and p=0.44 in validation cohort). Conclusion: A validated molecular-based integrative risk score was able to define subgroups of APL patients with different outcome independently of the Sanz score. The validation of the strong influence of the combination of molecular risk factors is particularly interesting as the new analyses of the three genes BAALC, ERG and WT1 separately did not confirm the strong results of the single analyses of each gene. The discrimination of patients according to the risk score is evident though. This argues for their integration in future studies. Figure 1 Comparison of the original cohort and the validation cohort. Discrimination by the integrative risk score (0-3points) for OS, RFS and CIR. Figure 1. Comparison of the original cohort and the validation cohort. Discrimination by the integrative risk score (0-3points) for OS, RFS and CIR. Disclosures No relevant conflicts of interest to declare.

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: Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on the easily accessible Sanz-Score, which combines leukocyte and platelet counts at initial diagnosis. This score showed significant differences in relapse-free survival (RFS) of APL patients in various studies and is currently used to determine whether a patient can be treated with ATRA and ATO alone or needs additional chemotherapy. However, to make therapeutic decisions based on a risk stratification system derived from the endpoint RFS bears the drawback that relapses are rare in APL and most events are deaths in complete remission (CR), which can be therapy related (e.g. toxicity). The cumulative incidence of relapse (CIR) therefore seems to be a better parameter for decision making with regard to therapy intensity. In this study, we optimized a risk score combining data on gene expression of BAALC (brain and acute leukemia, cytoplasmic), ERG (ets’ related gene) and WT1 (Wilms’ tumor 1) to retrospectively predict the CIR of APL patients. Methods: Data on BAALC, ERG and WT1 expression levels of 79 patients with newly diagnosed APL were obtained from bone marrow mononuclear cells using quantitative real-time RT-PCR in preceding studies. The following gene expression levels were identified as negative risk factors: BAALC expression ≥25th percentile (BAALChigh), ERG expression 〉75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low or high). As ERGhigh was the only independent predictor for relapse in multivariate analysis with a hazard ratio (HR) of 11.6, its predictive weight was regarded superior, respectively . Cut-off analyses were performed to determine the optimal ERG expression level cut-off for risk of relapse. Accordingly, the new cut-off for high ERG expression was set at ≥62nd percentile (optimized ERGhigh: optERGhigh; Sensitivity: 1.0, Specificity: 0.71). A combined 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, optERGhigh and WT1low or 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 two risk groups: 34 patients scored 0-1 points and 45 patients scored 2-3 points. CIR, overall survival (OS) and RFS were calculated using the Kaplan-Meier method and a log-rank test was used to compare differences between the two risk groups (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: Background Myelodysplastic Syndromes (MDS) are heterogeneous groups of hematopoietic malignancies that are highly susceptible to transformation into acute myeloid leukemia and clinically manifest with signs of severe anemia. Numerous studies have revealed recurring molecular alterations in hematopoietic stem/progenitor cells from MDS patients as presumable cause for the persistent dysfunction of their hematopoiesis. However, how these events translate into disturbed regulation of erythropoiesis is poorly understood so far. Characterization of the transcriptional network and associated epigenetic changes in dysplastic erythroprogenitor cells might therefore aid in the elucidation of functional molecular consequences finally leading to impaired erythropoiesis. Methods CD71+ erythroprogenitor cells were isolated from purified bone marrow cells via magnetic cell separation for 15 MDS patients (IPSS low/int-1 risk n=11, int-2/high risk n=4) and 7 age-adjusted healthy donors. Extracted DNA and RNA were processed and hybridized to Affymetrix Exon 1.0 ST Array and Illumina Infinium HumanMethylation450 Beadchips according to manufacturers’ instructions. Data analysis was carried out using Qlucore Omics Explorer 2.3 and in-house scripts for correlation of the two datasets were developed using Python 2.7.5. Results Using a false discovery rate/q-value of ≤ 4% as a cutoff for differential gene expression and CpG DNA methylation, both datasets allowed highly distinct clustering into MDS IPSS low/int-1 risk, int-2/high risk and healthy donor groups. In our MDS low/int-1 risk cohort 63 genes have been identified as significantly up- and 13 as downregulated (fold change ≥1.5, p≤0.001) while 64 genes showed up- and 28 downregulation in the MDS int-2/high risk group (fold change ≥1.5 p≤0.01). Surprisingly, global DNA methylation profiling revealed that 741 CpGs were detected as hypo- but only 19 as hypermethylated in low/int-1 risk, whereas 231 CpGs demonstrated hypo- and 479 hypermethylation in int-2/high risk MDS CD71+ cells relative to healthy controls (mean CpG methylation difference ≥10%, p≤0.0001). In order to discover genes susceptible to DNA methylation associated regulation of transcription, individual CpG methylation values were correlated with corresponding exon probeset expression intensities for every single gene. Using this approach, starting with 〉23x106 possible CpG/probeset combinations, 4418 displayed significant positive correlation, whereas only 2726 were negatively correlated (R≥0.6 or R≤-0.6, mean methylation difference MDS vs. healthy ≥5%, p≤0.01). Consequently, in MDS low/int-1 risk we identified strong hypomethylation as putative cause for a 5.8-fold upregulation of GDF15, an important regulatory factor involved in iron homeostasis. Moreover, DNA hypermethylation associated 6.6-fold knockdown of the transcription factor GTSF1, which has been associated with increased apoptosis in gametogenesis, was demonstrated for MDS int-2/high-risk. In this cohort, we also observed a 3-fold upregulation of LY6E, which has been shown to result in a strong block of differentiation and maintenance of self-renewal in avian erythroprogenitor cells. Consistently, gene set enrichment analysis identified a stem cell like gene signature as highly enriched in MDS CD71+ BM cells but also gene sets involved in oxidative phosphorylation as well as regulation of cell cycle and apoptosis. Conclusion Our integrative approach reveals novel candidate genes implicated in disturbed erythropoiesis in MDS and allows distinctive separation between healthy donors and MDS risk groups by assessment of epigenomic and transcriptomic landscapes derived from CD71+ bone marrow cells. DNA hypomethylation induced gene upregulation surprisingly appears to be a common event in MDS erythropoiesis which co-occurs with DNA hypermethylation induced gene silencing. In addition, frequent detection of significant positive correlation between DNA methylation and gene expression might add an additional layer of complexity to the current dogma of epigenetic gene regulation. Finally, the distinctively hypermethylated geneset in MDS high-risk as compared to the unexpected global hypomethylation phenotype in MDS low-risk might suggest a mechanistic explanation for the selective efficacy of demethylating substances specifically in the higher risk patient groups. Disclosures: No relevant conflicts of interest to declare.