BackgroundAlterations and senescence in bone marrow mesenchymal stromal cells of multiple myeloma patients (MM-BMMSCs) have become an important research focus. However the role of senescence in the pathophysiology of MM is not clear.MethodsCorrelation between senescence, cell cycle and microRNA expression of MM-BMMSCs (n = 89) was analyzed. Gene expression analysis, copy number analysis and methylation specific PCR were performed by Real-Time PCR. Furthermore, cyclin E1, cyclin D1, p16 and p21 genes were analyzed at the protein level using ELISA. Cell cycle and senescence were analyzed by FACS. MiRNA transfection was performed with miR-485-5p inhibitor and mimic followed by downstream analysis of senescence and cell cycle characteristics of MM-BMMSCs. Results were analyzed by Mann–Whitney U test, Wilcoxon signed-rank test and paired t-test depending on the experimental set up.ResultsMM-BMMSCs displayed increased senescence associated β-galactosidase activity (SA-βGalA), cell cycle arrest in S phase and overexpression of microRNAs. The overexpressed microRNAs miR-485-5p and miR-519d are located on DLK1-DIO3 and C19MC, respectively. Analyses revealed copy number accumulation and hypomethylation of both clusters. KMS12-PE myeloma cells decreased SA-βGalA and influenced cell cycle characteristics of MM-BMMSCs. MiR-485-5p was significantly decreased in co-cultured MM-BMMSCs in connection with an increased methylation of DLK1-DIO3. Modification of miR-485-5p levels using microRNA mimic or inhibitor altered senescence and cell cycle characteristics of MM-BMMSCs.ConclusionsHere, we show for the first time that MM-BMMSCs have aberrant methylation and copy number of the DLK1-DIO3 and C19MC genomic region. Furthermore, this is the first study pointing that multiple myeloma cells in vitro reduce both the senescence phenotype of MM-BMMSCs and the expression of miR-223 and miR-485-5p. Thus, it is questionable whether senescence of MM-BMMSCs plays a pathological role in active multiple myeloma or is more important when cell interaction with myeloma cells is inhibited. Furthermore, we found that MiR-485-5p, which is located on the DLK1-DIO3 cluster, seems to participate in the regulation of senescence status and cell cycle characteristics of MM-BMMSCs. Thus, further exploration of the microRNAs of DLK1-DIO3 could provide further insights into the origin of the senescence state and its reversal in MM-BMMSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1078-3) contains supplementary material, which is available to authorized users.

Mutation of the FMS-like tyrosine kinase-3 (FLT3) gene occurs with a frequency of 20-25% in acute myeloid leukemia (AML). Different studies have reported conflicting results, stating the importance of the length, position and number of internal tandem duplications (ITDs) for prognostic significance. In the present study, FLT3-ITD mutations were found in 51 (23%) of 218 patients with AML. Using sequence analysis we categorized ITD integration sites according to functional regions of the FLT3 receptor. Median ITD size was 61 bp. The insertion site was strongly correlated with ITD size: more C-terminal located inserted fragments were significantly bigger. Our data confirm that FLT3-ITD mutations identify a subset of young patients with AML with normal cytogenetics but with inferior outcome. Patients with AML with mutation localization outside the juxtamembrane domain showed no correlation with worse prognosis. A high mutant/wild-type ratio appears to have a major impact on the prognostic relevance.

Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Recently, microRNAs have become an important point of interest for research on micro-environmental interactions in MM with some evidence of tumor supportive roles in MM. In this study, we examined the role of miR-223 for MM support in BMMSCs of 56 patients with MM (MM-BMMSCs). miR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner compared to mono-cultured MM-BMMSCs. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2, or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs. In addition, reduction of miR-223 degraded the osteogenic differentiation potential of MM-BMMSCs. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced expression of VEGF and IL-6 by both cell types. These data provide first evidence that miR-223 participates in different MM supporting pathways in MM-BMMSCs inlcuding regulation of cytokine secretion and expression as well as osteogenic differentiation of MM-BMMSCs. More insights on the role of miR-223 in MM-BMMSCs and in cellular interactions between MM cells and MM-BMMSCs could provide starting points for a more efficient anti-myeloma treatment by targeting of notch signaling. © 2015 Wiley Periodicals, Inc.

BackgroundMutations in epigenetic modifiers were reported in patients with acute myeloid leukaemia (AML) including mutations in DNA methyltransferase 3A gene (DNMT3A) in 20%-30% patients and mutations in isocitrate dehydrogenase 1/2 gene (IDH1/2) in 5%-15% patients. Novel studies have shown that mutations in DNMT3A and IDH1/2 influence prognosis, indicating an increasing need to detect these mutations during routine laboratory analysis. DNA sequencing for the identification of these mutations is time-consuming and cost-intensive. This study aimed to establish rapid screening tests to identify mutations in DNMT3A and IDH1/2 that could be applied in routine laboratory procedures and that could influence initial patient management.MethodsIn this study we developed an endonuclease restriction method to identify the most common DNMT3A mutation (R882H) and an amplification-refractory mutation system (ARMS) to analyse IDH2 R140Q mutations. Furthermore, we compared these methods with HRM analysis and evaluated the latter for the detection of IDH1 mutations.ResultsOf 230 samples from patients with AML 30 (13%) samples had DNMT3A mutations, 16 (7%) samples had IDH2 R140Q mutations and 36 (16%) samples had IDH1 mutations. Sensitivity assays performed using serial dilutions of mutated DNA showed that ARMS analysis had a sensitivity of 4.5%, endonuclease restriction had a sensitivity of 0.05% and HRM analysis had a sensitivity of 5.9%–7.8% for detecting different mutations. HRM analysis was the best screening method to determine the heterogeneity of IDH1 mutations. Furthermore, for the identification of mutations in IDH2 and DNMT3A, endonuclease restriction and ARMS methods showed a perfect concordance (100%) with Sanger sequencing while HRM analysis showed a near-perfect concordance (approximately 98%).ConclusionOur study suggested that all the developed methods were rapid, specific and easy to use and interpret. HRM analysis is the most timesaving and cost-efficient method to rapidly screen all the 3 genes at diagnosis in samples obtained from patients with AML. Endonuclease restriction and ARMS assays can be used separately or in combination with HRM analysis to obtain more reliable results. We propose that early screening of mutations in patients with AML having normal karyotype could facilitate risk stratification and improve treatment options.

Acute myeloid leukemia (AML) is a complex disease caused by deregulation of multiple signaling pathways. Mutations in class III receptor tyrosine kinases (RTKs) have been implicated in alteration of cell signals concerning the growth and differentiation of leukemic cells. Point mutations, insertions, or deletions of RTKs as well as chromosomal translocations induce constitutive activation of the receptor, leading to uncontrolled proliferation of undifferentiated myeloid blasts. Aberrations can occur in all domains of RTKs causing either the ligand-independent activation or mimicking the activated conformation. The World Health Organization recommended including RTK mutations in the AML classification since their detection in routine laboratory diagnostics is a major factor for prognostic stratification of patients. Polymerase chain reaction (PCR)–based methods are well-validated for the detection of fms-related tyrosine kinase 3 (FLT3) mutations and can easily be applied for other RTKs. However, when methodological limitations are reached, accessory techniques can be applied. For a higher resolution and more quantitative approach compared to agarose gel electrophoresis, PCR fragments can be separated by capillary electrophoresis. Furthermore, high-resolution melting and denaturing high-pressure liquid chromatography are reliable presequencing screening methods that reduce the sample amount for Sanger sequencing. Because traditional DNA sequencing is time-consuming, next-generation sequencing (NGS) is an innovative modern possibility to analyze a high amount of samples simultaneously in a short period of time. At present, standardized procedures for NGS are not established, but when this barrier is resolved, it will provide a new platform for rapid and reliable laboratory diagnostic of RTK mutations in patients with AML. In this article, the biological and physiological role of RTK mutations in AML as well as possible laboratory methods for their detection will be reviewed.