ALBERT

Description: There is a variety of antineoplastic drugs that are based on natural compounds from ecological niches with high evolutionary pressure. We used two cell lines (Jurkat J16 and Ramos) in a screening to assess 300 different naturally occurring compounds with regard to their antineoplastic activity. The results of the compounds 4,6-dibromo-2-(2′,4′-dibromophenoxy)phenol (P01F03), 4,5,6-tribromo-2-(2′,4′-dibromophenoxy)phenol (P01F08), and 5-epi-nakijinone Q (P03F03) prompted us to perform further research. Using viability and apoptosis assays on the cell lines of primary human leukemic and normal hematopoietic cells, we found that P01F08 induced apoptosis in the cell lines at IC50 values between 1.61 and 2.95 μM after 72 h. IC50 values of peripheral blood mononuclear cells (PBMNCs) from healthy donors were higher, demonstrating that the cytotoxicity in the cell lines reached 50%, while normal PBMNCs were hardly affected. The colony-forming unit assay showed that the hematopoietic progenitor cells were not significantly affected in their growth by P01F08 at a concentration of 3 μM. P01F08 showed a 3.2-fold lower IC50 value in primary leukemic cells [acute myeloid leukemia (AML)] compared to the PBMNC of healthy donors. We could confirm the antineoplastic effect of 5-epi-nakijinone Q (P03F03) on the cell lines via the induction of apoptosis but noted a similarly strong cytotoxic effect on normal PBMNCs.

Description: Introduction: BRCA1 plays a key role in maintaining genomic stability and interacts directly with several proteins that regulate hematopoietic stem cell functions and are part of the Fanconi anemia (FA) double-strand break (DSB) repair pathway. Loss of BRCA1 in murine bone marrow causes hematopoietic defects similar to those seen in FA (Vasanthakumar, Blood 2016). BRCA1 is highly expressed in hematopoietic tissues, whereas its expression is lost in CML (Deutsch et al, Blood 2003) and t-AML (Scardocci et al, Br J Cancer 2006). Since FA is associated with an increased risk of developing myelodysplastic syndromes (MDS), we examined the protein expression of BRCA1 and three other important components of the FA DNA repair pathway, as well as PARP-1, whose co-alteration with BRCA causes synthetic lethality. Methods: We recently established a tissue microarray (TMA) with bone marrow biopsies from 119 MDS patients, 40 AML patients, and 11 normal controls. Biopsy material was retrieved from the Institute of Pathology, Heinrich-Heine-University Düsseldorf, Germany. The TMA was produced at SciLifeLab Tissue Profiling Facility in Uppsala, Sweden. Immunohistochemistry (IHC) protocols were established in our lab for the detection of BRCA1, BRCA2, FANCD2, H2AX, and PARP1. Semi-quantitative analysis was done according to Remmele-Stegner immunoreactive score (IRS) with a point system from 0 to 12. Expression was categorized as absent (0-2), weak (3-4), moderate (5-8) or strong (9-12). For correlating protein expression with clinical data from the Düsseldorf MDS Registry, samples were dichotomized into 'low expression' (1-4) and 'higher expression' (5-12). Prognostic analysis was restricted to 80 MDS patients who did not receive disease-modifying therapy. Results: Similar to normal controls, most patients with AML showed strong expression of the proteins under consideration (Fig. 1). In contrast, low expression was found in about 60% of MDS patients. Most MDS patients showed concordant, dichotomous expression of all five proteins (either all up or all down). Correspondingly, the protein expression landscapes look similar (Fig. 1). Almost perfect concordance was noted for PARP1 and H2AX. Low expression of FA/BRCA pathway proteins was not correlated with MDS type (WHO 2008) or IPSS-R risk group. Patients with higher expression of at least 3 of the 5 proteins survived longer (45 vs. 26 months) and had a longer time to AML development (median not reached at 180 months vs. 53 months), but the differences did not reach statistical significance (p=0.108 and p=0.159, respectively). Interestingly, patients with low expression of PARP1 were significantly more likely to show any chromosomal aberration (p=0.02) or an unfavourable karyotype (intermediate, high-risk or very high risk according to IPSS-R) (p=0.016). The same was true for patients with low expression of H2AX (p=0.013 and p=0.01). Conclusions: This is the first TMA-based investigation of FA/BRCA pathway protein expression in MDS. In stark contrast to AML, 60% of MDS patients showed low protein expression in a concordant manner. This could reflect synexpression of genes that share common cis- and trans-acting control elements. Alternatively, it could be the result of aberrant splicing, since at least 50% of MDS patients have spliceosome mutations that render a large spectrum of messenger RNAs susceptible to nonsense-mediated decay. Abnormal splicing factors may also influence the stability (and thus expression) of certain proteins through abnormal protein-protein interactions. Further IHC analyses will show whether the dichotomous protein synexpression pattern we observed in MDS extends to proteins that are unrelated to DNA maintenance. Underexpression of FA/BRCA pathway proteins may cause chromosomal instability, as suggested by our finding of significantly more frequent karyotype anomalies in patients with low PARP1 or H2AX expression. This is in accordance with the known role of H2AX as suppressor of oncogenic chromosome translocations, and with an accelerating effect of PARP1 deficiency on centrosome amplification in BRCA1-deficient cells. FA/BRCA pathway protein underexpression may not only contribute to a better understanding of MDS versus AML pathogenesis, but may also have therapeutic implications, e.g. by affecting the response to treatment with hypomethylating agents, which are known to promote DNA double strand breaks. Figure 1 Figure 1. Disclosures Gattermann: Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accomodation expenses, Research Funding.

Description: During the last decade, chronic myeloid leukemia (CML) has been mainly characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the protooncogene BCR-ABL. This constitutively active tyrosine kinase is widely considered as the cause of the disease. Even though BCR-ABL transcripts are found in every dividing hematopoietic cell and thus, the disease is likely to originate from a primitive stem cell, the “cell of origin” is still a matter of debate. Despite the active “leukemia stem cell” discussion, very few characteristics of the “cancer stem cell” are established to date. In order to get further molecular insights into CML stem and progenitor cells, we examined CD34+ cell subsets obtained from bone marrow of 7 patients with CML in chronic phase in comparison with 5 healthy volunteers. CD34+ cells were immunomagnetically selected and high-speed cell sorting of lineage-negative, CD34+, CD38−, hematopoietic stem cells and myeloid progenitors was performed. Progenitors were further subdivided by anti-IL-3Ralpha and anti-CD45RA staining. Following RNA extraction, a two-cycle amplification procedure was used to generate cDNA for the hybridization with Affymetrix U133A2.0 arrays. After performing smoothening spline normalization, we applied the perfect match-mismatch difference model algorithm to calculate expression values (dChip). Hierarchical cluster analysis was performed using a correlation based centroid linkage algorithm. Hereby we could discriminate the HSCs, CMPs, and MEP subsets. Corroboration of RNA expression was performed by real-time RT-PCR for selected genes. Comparing the HSC subsets of CML patients with healthy controls we found 98 differentially expressed genes. 87 genes had a lower expression level in CML HSCs whereas 11 genes had a higher one. Among the downregulated genes in CML were transcriptions factors involved in myelogenesis and proliferation and several adhesion molecules associated with homing and migration of the HSCs. On the other hand, the Leptin receptor and BCR-ABL downstream targets were found to be upregulated. Within the common myeloid progenitor (CMP) compartment 37 genes were significantly differentially regulated. Twenty genes had a higher expression level in CML CMPs, 17 genes were downlegulated. Hematopoietic cell-specific cell cycle inhibitor MS4A3 was among the significantly downregulated genes whereas genes of the retinoblastoma and E2F families as well as inhibitors of the Wnt-signaling pathway were upregulated. Looking at megakaryocte-erythrocyte progenitors (MEP) in CML, key mediators of G2-M cell cycle transition were downregulated indicating a lower proliferative capacity of this subset. No transcriptional differences have been observed between granulocyte-macrophage progenitors from CML patients and healthy volunteers. Interestingly, among all other subsets myeloperoxidase (MPO) was downregulated in the CML samples and the Leptin receptor was upregulated. Our results provide novel insights into the biology of CML and potentially provide the basis for the characterization of a candidate CML stem cell.

Description: Multiple myeloma (MM) is a clonal plasma cell disorder frequently accompanied by hematopoietic impairment. We show that hematopoietic stem and progenitor cells (HSPCs), in particular megakaryocyte-erythrocyte progenitors, are diminished in the BM of MM patients. Genomic profiling of HSPC subsets revealed deregulations of signaling cascades, most notably TGFβ signaling, and pathways involved in cytoskeletal organization, migration, adhesion, and cell-cycle regulation in the patients. Functionally, proliferation, colony formation, and long-term self-renewal were impaired as a consequence of activated TGFβ signaling. In accordance, TGFβ levels in the BM extracellular fluid were elevated and mesenchymal stromal cells (MSCs) had a reduced capacity to support long-term hematopoiesis of HSPCs that completely recovered on blockade of TGFβ signaling. Furthermore, we found defective actin assembly and down-regulation of the adhesion receptor CD44 in MM HSPCs functionally reflected by impaired migration and adhesion. Still, transplantation into myeloma-free NOG mice revealed even enhanced engraftment and normal differentiation capacities of MM HSPCs, which underlines that functional impairment of HSPCs depends on MM-related microenvironmental cues and is reversible. Taken together, these data implicate that hematopoietic suppression in MM emerges from the HSPCs as a result of MM-related microenvironmental alterations.

Description: Pancytopenia is the most prominent clinical finding in patients with acute myeloid leukemia (AML) and represents a major cause for morbidity and mortality. So far, the underlying mechanisms leading to hematopoietic insufficiency in AML are poorly understood and therefore often mechanistically summarized as marrow replacement by infiltrating leukemic cells. Mesenchymal stem and progenitor cells (MSPC) are integral components of the bone marrow (BM) microenvironment and play an indispensable role for the regulation of normal hematopoiesis. Two AML mouse models have recently shown, that expansion of the leukemic clone leads to numeric changes and functional disturbances of niche components such as MSPC and osteoblasts, resulting in insufficient hematopoietic support (Hanoun et al. 2014; Frisch et al. 2012). As the knowledge about MSPC in human AML is limited so far, we conducted a detailed analysis of AML-derived MSPC in order to elucidate their contribution to hematopoietic failure. For this purpose we investigated the molecular and functional properties of BM-derived MSPC of 46 patients with AML covering all relevant subtypes according to WHO classification at diagnosis and/or during course of disease and compared them with MSPC functions of healthy controls. Hematopoietic insufficiency in the 31 patients at diagnosis was mirrored by 76% of the patients having bi- or pancytopenia and by median ANC of 1183/μl, median hemoglobin of 9.0 g/dl, and median platelet count of 67.000/μl. MSPC of these newly diagnosed patients exhibited significantly impaired growth capacities as shown by an altered morphology, reduced CFU-F activity, a lower number of passages and cumulative population doublings. While adipogenic differentiation potential was not affected, osteogenic differentiation potential of AML-derived MSPC was significantly reduced as indicated by cytochemical stainings, reduced Osterix and Osteocalcin (OC) mRNA levels as well as OC serum levels. Furthermore we detected altered mRNA and/or protein expression of key molecules involved in the regulation of hematopoietic stem and progenitor cells (HSPC), namely SCF, Angiopoietin-1, Jagged1 and Osteopontin. Functionally, this translated into a significantly diminished ability of AML-derived MSPC to support healthy CD34+ HSPC in LTC-IC assays. This insufficient stromal support was reversible and correlated with disease status, as LTC-IC frequency returned to normal values in patients in remission, but remained low in patients with refractory disease. Along with this we also observed a significant increase of OC serum levels in patients, who achieved complete remission. These data clearly suggests a direct causal relationship between the presence of leukemic cells and MSPC functionality. In further support of this idea we observed reduced proliferation and osteogenic differentiation of healthy MSPC following cultivation in conditioned media (CM) of 4 AML cell lines (THP-1, HL-60, MV4-11, MOLM-13). A comparable inhibitory effect of AML cells on healthy MSPC growth was seen in transwell-assays arguing in favor for a cell-contact independent mechanism. In summary, our data show that AML-derived MSPC are structurally and functionally altered resulting in an insufficient stromal support for normal hematopoiesis in AML. The correlation between clinical remission status and stromal support function together with the finding, that healthy MSC can adopt an AML-like phenotype when exposed to AML-CM suggest an instructive role of the leukemic precursor cells. Disclosures Dührsen: Celgene: Honoraria, Research Funding. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Novartis: Research Funding; Celgene: Honoraria, Research Funding; AMGEN: Research Funding; Janssen-Cilag: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria. Kobbe:Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medac: Other; Astellas: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Neovii: Other.

Description: Abstract 598 Thrombocytopenia is prevalent in up to 65% of patients with myelodysplastic syndrome (MDS) at the time of diagnosis and thrombocytopenic hemorrhage is a significant clinical problem that is often complicated by platelet aggregation defects. Little is known about the pathophysiology of this insufficient platelet function. Here, we delineate a reduced expression of critical platelet aggregation-related proteins by analyzing the platelet proteome of 7 patients with MDS and 7 normal donors. Patients' median platelet count was 60 × 10E9/L (range 37–109 × 109/L) and none of the patients examined had received prior anticoagulant treatment, chemotherapy or platelet transfusions. Differential 2-dimensional in-gel electrophoresis coupled with a time-of-flight Ultraflex-Tof/Tof mass spectrometer enabled the discovery of 120 differential protein spots. Among these, we identified a total of 35 proteins including 26 proteins that are integral part of the integrin aIIbβ3 receptor (GPIIb/IIIa, Fibrinogen receptor) signaling such as Talin-1 and Vinculin. In resting platelets the integrin aIIbβ3 receptor exhibits a low-affinity (inactive) state which is shifted to a high-affinity (active) state following inside-out activation. Talin-1 expression has been shown to be essential for this inside-out activation of the integrin aIIbβ3 receptor and consecutive platelet aggregation in an in-vivo model. We hypothesized that the reduced expression of Talin-1 and its co-factor Vinculin may inhibit activation of the integrin aIIbβ3 receptor and thereby contribute to the platelet aggregation defect seen in patients with MDS. Therefore, we performed further functional studies on integrin aIIbβ3 receptor activation and platelet spreading/aggregation with platelets derived from an independent cohort of 7 patients with MDS and 7 normal donors. In this new cohort, patients' median platelet count was 94 × 109/L (range 60–120 × 109/L) and again all patients had never received prior platelet transfusions or anti-coagulant treatment. When we looked at the surface expression of the integrin aIIbβ3 receptor on resting platelets by means of flow-cytometry, we did not detect any differences between platelets from patients with MDS and normal donors. Then, we activated platelets from normal donors and patients with MDS with 0.01U/μl and 0.001U/μl thrombin and measured binding of PAC-1, which is highly specific for detection of the active form of the integrin aIIbβ3 receptor. Here, we found a significantly lower shift from the inactive to the active form in platelets derived from patients with MDS dropping from 92.15% and 91.46% in normal donors to 41.97% and 48.45% (p = 0.01 and p = 0.006), respectively. We confirmed this suggested lack of adhesion and aggregation capacities in MDS platelets by confocal microscopy and single platelet imaging of washed platelets stimulated with 0.01U/μl thrombin which were adhered to immobilized fibrinogen. Consecutive platelet aggregation assays also revealed an insufficient response to stimuli like Collagen and ADP. Our findings provide for the first-time insight into the molecular pathology of defective platelet aggregation in MDS and suggest a mechanism of defective inside-out signaling caused by a reduced expression of proteins required for integrin aIIbβ3 receptor activation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1487 Poster Board I-510 TIMP–1 protein (Tissue Inhibitor of Metalloproteinases) is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem cell homing and mobilization. We first screened gene array data sets of CD34+ human hematopoietic stem and progenitor cells (HSPCs) and found TIMP–1 mRNA expression. In this study we show that TIMP–1 co-localizes with the tetraspanin CD63 and Beta-1-Integrin. Furthermore, we found a functional interaction of TIMP-1 with its receptor complex on G-CSF mobilized HSPCs. All experiments were carried out using highly enriched CD34+ cells. Co-immunoprecipitation shows that TIMP–1 binds to CD63. Using high resolution Stimulated Emission Depletion (STED) microscopy we could confirm co-localization of TIMP-1 and CD63 as well as Beta-1 Integrin and CD63. To further characterize the role of TIMP-1 in the Beta-1-Integrin signaling, we used an antibody specific to the active form of Beta-1- Integrin. Flow cytometric analysis revealed a significantly higher number of active Beta-1-Integrin in TIMP-1 stimulated cells suggesting TIMP-1 to activate the receptor complex on CD34+ cells. For functional analysis of the receptor complex formation, transwell migration assays were performed showing significantly increased migratory capacities of TIMP–1 treated cells. Additionally, TIMP–1 stimulation leads to a significantly increased adhesion rate of CD34+ cells to the fibronectin-coated dish. To assess a potential role of TIMP–1 in apoptosis, CD34+ HSPCs were co-incubated with thapsigargin and TIMP–1 or DMSO as a control. Subsequent flow cytometric analysis of cleaved Caspase-3 revealed a decrease of apoptotic cells in the TIMP-1 treated samples. In summary, we can show that TIMP-1, CD63 and Beta-1-Integrin form a complex on CD34+ HSPCs. TIMP-1 activates the Beta-1-Integrin signaling in HSPCs and alters the adhesive as well as the migratory behavior of CD34+ HSPCs. Furthermore, TIMP-1 induces an antiapoptotic effect in CD34+ cells. The functional effects of TIMP-1 in HSPCs might be of relevance in clinical hematopoietic stem cell transplantation so that we are currently about to verify these effects in an in vivo model. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

Description: Abstract 2620 Introduction: In our previous report (Zohren et al., Blood 2008) we could show that the blockade of the heterodimer VLA-4 by the monoclonal IgG4 antibody natalizumab leads to a significant increase in circulating CD34+ cells in patients with multiple sclerosis (MS). We now extend our analysis on the influence of natalizumab on CD34+ cells comparing bone marrow (BM) and peripheral blood (PB) derived CD34+ cells of natalizumab patients with those from healthy donors. Methods: A total of 83 patients with MS receiving natalizumab were included. In vitro adhesion, migration and apoptosis assays as well as LTC-IC of immunomagnetically enriched CD34+ cells were conducted. Flow cytometric analyses were performed to assess phenotype and composition of the CD34+ subsets. Results: The median concentration of circulating CD34+ cells was significantly greater compared to normal donors (7.7/μL vs. 1.8/μ L; p= 0.0001) and remained relatively stable during a one year treatment with natalizumab. Leukocyte cell counts, the number of T cell subsets as well as the number of CD19+ B cells and CD56+ natural killer cells were in normal range in PB and BM after short- and long-term treatment with natalizumab. However, we found significantly reduced adhesion and migration abilities of circulating CD34+ cells under natalizumab treatment in comparison to G-CSF mobilized CD34+ cells of healthy donors. Moreover, the self-renewal capacity of these cells was poor. In contrast, no significant difference was seen between the BM of natalizumab patients and the BM of healthy donors with regard to cellularity and proportion of CD34+ cells. In addition, neither co-expression of CD49d nor the adhesion ability of the BM derived CD34+ cells revealed a significant difference between the two collective. Conclusions: Our data indicate that natalizumab mediates an increase in circulating CD34+ cells by impaired homing. These findings argue against the use of natalizumab-exposed PB CD34+ cells for transplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2393 Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell disorders and research in this field has mainly focused on hematopoietic stem and progenitor cells (HSPC). Still, recent data from mouse models indicate that the bone marrow (BM) microenvironment might be involved in the pathogenesis MDS (Raaijmakers et al., 2010). The role of mesenchymal stromal cells (MSC) in particular as a key component of the BM microenvironment remains elusive in human MDS and data so far are controversial. Design/Methods: We therefore investigated MSC and immunomagnetically enriched CD34+ HSPC from BM of 42 untreated patients (pts) with MDS (12 RCMD, 12 RAEB, 12 sAML, 3 del5q, 1 CMML-1, 1 MDS hypocellular, 1 MDS unclassifiable according to WHO) and age-matched healthy controls (HC, n=13). MSC were examined with regard to growth kinetics, morphology and differential potential after isolation and expansion according standard procedures in line with the international consensus criteria (Dominici et al., 2006). Furthermore corresponding receptor-ligand pairs on MSC and CD34+ cells (Kitlg/c-kit; CXCL12/CXCR4; Jagged1/Notch1; Angpt1-1/Tie-2; ICAM1/LFA-1) were investigated by RT-PCR. Results: In MDS, the colony forming activity (CFU-F) of MSC was significantly reduced in comparison to HC (median number of colonies per 1×107MNC in MDS: 8, range 2–74 vs. HC: 175, 10–646, p=0.003) and this was also true when looking at the different subtypes (RCMD median: 16, p=0.04; RAEB median: 8, p=0.31; sAML median: 26, p=0.02). According to this, MSC from pts with RCMD and del5q could only be maintained in culture for a lower number of passages (median number of passages: MDS 3 passages, range 1–15; HD 14 passages, range 8–15, p=0.01), had a lower number of cumulative population doublings (CPD) and needed a longer timer to reach equivalent CPD (MDS median: 18,16 CPD, HD median: 33,68 CPD, p=0,0059). All types of MDS-MSC showed an abnormal morphology, while an impaired osteogenic differentiation potential was exclusively observed in pts with RCMD. These findings of an altered morphology together with a diminished growth and differentiation potential prompted us to test, whether the interaction between MSC and CD34+ HSPC in BM of pts with MDS was also disturbed. On the MDS-MSC, we found a significant lower expression of Angpt1 in pts with RAEB (3.5-fold, p=0.01) and del5q (4.9-fold, p=0.009) compared to HD. The expression of CXCL12 (2.5-fold, p=0.057) and jagged1 was reduced in trend in MSC from pts with MDS, while no differences were observed with regard to the expression of kitlg and ICAM1. When looking on CD34+ cells, we found a significantly reduced expression of CXCR4 (RCMD 2.5-fold, p=0.02; RAEB 2.46-fold, p=0.02), notch1 (RCMD 6-fold, p=0.04) and Tie-2 (RAEB 5.91-fold, p=0.02) in pts with MDS, while LFA-1 was overexpressed in pts with RAEB (2.6-fold, p=0.036). Conclusion: Taken together, our data indicate that MSC from pts with MDS are structurally altered and that the crosstalk between CD34+ HSPC and MSC in the BM microenvironment of pts with MDS might be deregulated as a result of an abnormal expression of relevant receptor-ligand pairs. Ongoing research is required to corroborate these findings and to definitely address their functional relevance for the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.