ABSTRACTtypes including MDS associated with single del(5q), and characterized their clonogenicity and differentiation potential. Methods Human samplesTwenty MDS patients were studied; their characteristics are detailed in Online Supplementary Table S1. Our control cohort consisted of one male and five female healthy individuals undergoing orthopedic surgery with a median age of 60 years (range, 56-65). MSC were obtained as previously described. 18 Patients were classified for the study as "lower risk" with and without del(5q) (LR, LR-5q ) (<5% bone marrow blasts and IPSS-low/int-1), and as "higher-risk" (HR) (>5% bone marrow blasts and IPSS-int-2/high). Magnetically sorted HSPC were obtained from peripheral blood of healthy individuals after mobilization. Samples were collected after approval by our Institutional Review Board, and written informed consent was obtained. A detailed description is available in the Online Supplementary Material. Lenalidomide treatmentLEN was kindly provided by Celgene (Munich, Germany) and prepared using 10% dimethyl-sulfoxide as a vehicle. The experiments were performed with 0.1 μM and 1 μM of the drug. Characterization of the mesenchymal stromal cellsClonality, self-renewal, generation of single cell-derived colonies (SCD), expression of cell surface and adhesion molecules with flow cytometry, differentiation potential, proliferation, viability, apoptosis, senescence, and cell cycle were studied. Detailed information about the experiments is provided in the Online Supplementary Material. Co-culture of myelodysplastic syndrome-mesenchymal stromal cells with hematopoietic stem and progenitor cells, clonogenicity and apoptosis assayThe capacity of MSC to support HSPC was tested using a 4-week cobblestone area-forming (CAF-C) assay and a 14-day colony-forming unit granulocyte-erythrocytemonocyte/macrophage (CFU-GEMM) assay as described in the Online Supplementary Material. HSPC or the cell line KG1-α were labeled with carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) prior to co-culture with MSC for 72 h, or left unlabeled and co-cultured for 24 h, with or without the addition of 100 nM recombinant tumor necrosis factor alpha (TNF-α, Peprotech). Apoptosis and proliferation were studied by annexin-V staining and CFSE dilution using flow cytometry. Cytokine measurement and transwell migration assaySupernatant from MSC was obtained after culture with or without LEN and used for enzyme-linked immunoabsorbent assay (ELISA) determinations of SDF-1α, angiopoietin-1 (ANG1), and stem cell factor (SCF) levels. Supernatant was also used for a 4-h transwell migration assay with 1x10 5 HSPC, as described previously. 18 The CXCR4 antagonist AMD3100 (Sigma) was used at a concentration of 10 μM.RNA extraction, complementary DNA synthesis and real time polymerase chain reaction mRNA extracted with Trizol reagent (Invitrogen) was transcribed into complementary-DNA and used for real time polymerase chain reaction (RT-PCR) analysis for fatty acid binding protein 4 (FABP4), SDF-1α , ANG1, and SCF. Ab...

Purpose: Retrospective, surrogate marker-based studies have found inconsistent associations between systemic iron overload (SIO) and adverse outcome in patients undergoing allogeneic stem cell transplantation (allo-SCT). As a consequence, the impact of SIO in this context remains under debate. The aim of this study was to test whether the objective pretransplant quantification of liver-iron content (LIC) by magnetic resonance imaging (MRI) could circumvent these limitations and conclusively define the prognostic relevance of SIO.Experimental Design: The correlation between pretransplant LIC and surrogate parameters as well as the impact of SIO on posttransplant outcome was assessed within an observational study of patients (n ¼ 88) with either myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) undergoing allo-SCT.Results: Ferritin levels of 1,000 ng/mL or more provided only poor specificity (31.8%) for predicting elevated LIC (!125 mmol/g) and even higher thresholds (!2,500 ng/mL) lacked an association with nonrelapse mortality (NRM). In contrast, LIC 125 mmol/g or more was a significant risk factor for NRM in uni-and multivariate analysis (HR ¼ 2.98; P ¼ 0.016). Multivariate Cox-regression further showed that LIC 125 mmol/g or more was associated with a decreased overall survival (HR ¼ 2.24, P ¼ 0.038), whereas ferritin or transfusion burden were not.Conclusions: SIO reflected by LIC is an independent negative prognostic factor for posttransplant outcome in patients with AML and MDS undergoing allo-SCT. Therefore, MRI-based LIC, and not interference-prone serum markers such as ferritin, should be preferred for pretransplant risk stratification and patient selection in future clinical trials.

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BackgroundThe M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia.MethodsSites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA.ResultsExpression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia.ConclusionsThe study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.