Mesenchymal stem cells from myelodysplastic syndromes are functionally and genomically abnormal
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE10822 | GEO | 2008/08/01
SECONDARY ACCESSION(S): PRJNA107425
REPOSITORIES: GEO
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Project description:Myelodysplastic syndromes (MDS) are a group of clonal disorders of hematopoietic stem cells. Mesenchymal stem cells (MSC) are the progenitors of the Bone Marrow (BM) stroma and have been involved in the physiopathology of MDS. The presence of cytogenetic aberrations on MSC from MDS patients is controversial. The aim of the study is to characterize BM derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridisation (FISH) analysis and genetic changes by array based comparative genomic hybridization (array-CGH). 32 cases of untreated MDS were included in the study. MSC from MDS achieved confluence at a slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was also lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic, and gains were more frequent than looses. The results of array-CGH were confirmed by FISH. When an unsupervised hierarchical cluster analysis was performed two clusters were identified: one of them included the 5q- syndrome patients, while the other incorporated the rest of the MDS patients. Our results shows, for the first time, that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to particular MDS subtypes. Keywords: Genomic comparison between mesenchymal cells 32 BM cells from âde novoâ MDS, corresponding to 29 patients, were included in the study. Male/female ratio was 14/15 and median age was 70 years (range 34-89). MDS diagnosis was established by peripheral blood counts and morphology, BM morphology and cytogenetic analyses and classified according to the WHO proposals. 12 Patientsâ characteristics are summarized in table 1. Three patients were included twice in the study: at the diagnosis of one RA (case 2) and two Unclassifiable MDS (cases 29,30), and at the progression into RAEB-2 (cases 16, 20 and 18). None of them had received treatment during this period of time, other than supportive therapy. Normal BM samples were obtained from 15 healthy donors, 8 males and 7 females, with a median age of 57 (range 37-88). Written informed consent was obtained from all patients and donors according to ethical guidelines of our institution. Genomic-wide analysis of DNA copy number changes of MSC from 13 cases was performed using array-CGH. Slides containing 3032 BACs were produced in âCentro de Investigación del Cáncerâ (Salamanca, Spain). The particular BAC and P-1 derived artificial chromosome set used to produce this array is distributed to academic institutions by the Welcome Trust Sanger Institute (Cambridge, United Kingdom) and contains targets spaced at ï» 1 Mb density over full genome, a set of subtelomeric sequences for each chromosome arm, and a few hundred probes selected for their involvement in oncogenesis. The clone content is available in the âCytoviewâ windows of the Sanger Institute mapping database site, Ensembl (http://www.ensembl.org/). According to this database, clones were ordered along the chromosomes. These clones were isolated from their bacterial cultures with the relevant antibiotics and the DNA was extracted with standard protocol Welcome Trust Sanger Institute (Cambridge, UK). 10 ng of DNA (BAC/PAC) was used as a template for three DOP-PC. 22;23 These products were ethanol precipitated and dissolved in distilled water. A minimum of three replicates per clone were printed by Microgrid II (Biorobotics) on each slide (Ultragaps Coated Slides, Corning) using the aqueous dimethylsulfoxide buffer as spotting solution. Briefly, for labelling reactions, 1 μg of nonamplified genomic DNA, test and reference were digested separately with DpnII restriction enzyme (New England Biolabs, Beverly, MA). For microarray hybridization, the digested DNAs were separately labelled using random primers (Bioprimer labelling kit, Invitrogen) and cyanine dye-3-dCTP and cyanine dye-5-dCTP fluorescent dye to paired hybridisation samples (Amersham Biosciences). The incorporated of the label nucleotide was quantified using the Nanodrop spectrophotometer. Labelled test and reference DNAs were mixed equitably, co-precipitated in presence of Cot-1 human DNA (Roche, Indianapolis, IN) with ethanol, washed, and resuspended in hybridization solution (50% Formamide, 10% Dextran sulfate, 2X standard saline citrate, 10 mM Tris pH 7.6, 2.7% sodium dodecyl sulfate and 10 μg/ μl of yeast tRNA). DNA mixtures were hybridized to the arrays in the TECAN HS4800 Pro according to the manufacturerâs recommended protocol. Images and signal intensities were acquired using GenePix4000B (Axon Instruments, Burlingame, CA) dual laser scanner in combination with GenePixPro4.0 (Axon Instruments) imaging software. Image and Data analysis. Spot intensities measured by the GenePix Pro 4.1 software (Axon Instruments, Wetsbur BV, Leuden, The Netherland). Pixel intensities for each feature were integrated and median values were determined, and the local background was calculated. For each spot the intensities were corrected by subtracting the local background for both wavelengths, and only spots with signal intensities al least threefold above background signal intensities were included in the analysis. The median of the ratios of all spots was calculated and used to normalize all data points. From each feature, the log2 value of the average of the normalization ratios of the spots was calculated and used to display the data. In addition, fluorescence ratios were normalized for each microarray using the print-tip loess method and the background subtraction with the Diagnosis and Normalization Arrays Data tool. 24 The cut-off level was determined for each individual experiment after avering the ratios from the two color-swith hybridizations and subsequent for normalization, and calculate the mean and Standard derivations to define the cut-off level as means plus/minus two times the standard derivations. The clones that showed possible changes were submitted to the Database of Genomics Variants (http://projects.tcag.ca/variation/) and chromosomal Imbalances and Phenotype in Humans using Ensembl Resources (DECIPCHER: http: www sanger. ac.uk /PostGenomics/decipher/. Clustering Unsupervised clustering We converted the relative ratio value for each BAC clones in each sample to score of 1 (gain/amplified), 0 (no change), or -1 (loss) based on the 2 SD standards described above and analyzed with Cluster and TreeView) 25 (Cluster and TreeView software) based on the average linkage method with the centered correlation metric was used (or Pearson Uncentered). Supervised Sam analysis was used http://www-stat.stanford.edu/ tibs/SAM) 26 to identify BACs that segregate subgroups on MSC. BACs with an SD of zero across the data set before analysis were removed.
2010-05-25 | E-GEOD-10822 | biostudies-arrayexpress
Project description:Isolated deletions of the long arm of chromosome 5 (del(5q)) are observed in 10% of myelodysplastic syndromes (MDS) and are associated with a more favorable prognosis, although the clinical course varies considerably. If one or more additional chromosomal aberration/s are present this correlates with a significant shorter overall survival. To assess the frequency of hidden abnormalities in cases with an isolated cytogenetic del(5q), we have performed a genome wide high resolution 44K 60mer oligonucleotide array CGH study using DNA from bone marrow cells of 12 MDS and one AML patient. Additional chromosomal aberrations were identified in three of 13 cases (23%): in one case, a single additional hidden 5.6 Mb deletion of 13q14 and in two cases, multiple larger aberrations involving many chromosomes. Fluorescence in situ hybridization (FISH) demonstrated that aberrations present in only 10% of the bone marrow cells were detectable by aCGH. Furthermore with oligonucleotide aCGH the deletion end points in 5q were mapped precisely, revealing a cluster of proximal break points in band q14.3 (n=8) and a distal cluster between bands q33.2 to q34 (n=11). This study shows the high resolution of oligonucleotide CGH arrays for precisely mapping genomic alterations and for refinement of deletion end points. In addition the high sensitivity of this method enables the study of whole bone marrow cells from MDS patients, a disease with a low blast count. Future studies of more patients with isolated del(5q) for hidden abnormalities will be necessary to evaluate the impact of these on the variable prognosis in these patients and to further define new genetic subgroups. Keywords: Myelodysplastic syndromes, MDS, array CGH, del(5q), hidden aberrations
2007-09-11 | GSE8804 | GEO
Project description:PURPOSE: Bone marrow (BM) is a common homing organ for early disseminated tumor cells (DTC) and their presence can predict the subsequent occurrence of overt metastasis and survival in lung cancer. It is still unclear whether the shedding of DTC from the primary tumor is a random process or a selective release driven by a specific genomic pattern. EXPERIMENTAL DESIGN: DTCs were identified in BM from lung cancer patients by an immunocytochemical cytokeratin assay. Genomic aberrations and expression profiles of the respective primary tumors were assessed by microarrays and FISH analyses. The most significant results were validated on an independent set of primary lung tumors and brain metastases. RESULTS: Combination of DNA copy number profiles (array CGH) with gene expression profiles identified five chromosomal regions differentiating BM-negative from BM-positive patients (4q12-q32, 10p12-p11, 10q21-q22, 17q21 and 20q11-q13). Copy number changes of 4q12-q32 were the most prominent finding, containing the highest number of differentially expressed genes irrespective of chromosomal size (p=0.018). FISH analyses on further primary lung tumor samples confirmed the association between loss of 4q and the BM-positive status. In BM-positive patients 4q was frequently lost (37% vs. 7%), whereas gains could be commonly found among BM-negative patients (7% vs. 17%). The same loss was also found to be common in brain metastases from both small and non-small-cell lung cancer patients (39%). CONCLUSIONS: Thus, our data indicates for the first time that early hematogeneous dissemination of tumor cells might be driven by a specific pattern of genomic changes.
2009-10-05 | GSE13191 | GEO
Project description:Abnormalities in DNA copy number are frequently found in patients with multiple anomaly syndromes and mental retardation. Array-CGH is a high resolution whole-genome technology which improves detection of submicroscopic aberrations underlying these syndromes. Eight patients with mental disability, multiple congenital anomalies and dysmorphic features were screened for submicroscopic chromosomal imbalances using the GenoSensor Array 300 Chip. Subtelomeric aberrations previously detected by FISH analysis were confirmed in two patients, and accurate diagnosis was provided in two previously undiagnosed complex cases. Microdeletions at 15q11.2-q13 in a newborn with hypotonia, cryptorchidsm and hypopigmentation were detected with few discrepancies between the array results and FISH analysis. Contiguous microdeletion of GSCL, HIRA and TBX1 genes at 22q11.2 was identified in a previously undiagnosed boy with an unusual presentation of the VCF/DiGeorge spectrum. In a newborn with aniridia, a borderline false negative WT1 deletion was observed, most probably because of differences between the size of the genomic deletion and the microarray probe. A false positive rate of 0.2% was calculated for clone-by-clone analysis, while the per patient false positive rate was 20%. Array-based CGH is a powerful tool for the rapid and accurate detection of genetic disorders associated with copy number abnormalities, and can significantly improve clinical genetic diagnosis and care. Keywords: comparative genome hybridization (CGH)
2006-07-28 | GSE4775 | GEO
Project description:Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease with a variable clinical course strictly dependent on cytogenetic and molecular features. However, in 15-20% of cases both conventional cytogenetic and FISH analyses do not show any kind of abnormality. With the aim to identify dependable molecular prognostic factors in this subgroup, we evaluated 171 CLL patients, without aberrations detected by chromosome banding and FISH analysis. A comprehensive analysis was performed including genomic arrays (CGH+SNP), IGHV status, flow cytometry and a targeted sequencing. By genomic arrays, we detected 73 aberrations in 39 patients (23%). Most frequently, patients had 1 aberration (25/171; 15%), while 14 patients (8%) had at least 2 aberrations. IGHV unmutated status was present in 53/171 (31%) patients. SF3B1 was the most frequently mutated gene (26/171 patients; 15%), followed by NOTCH1 (n=15, 9%), ATM (n=5; 3%); TP53 (n=5; 3%); KLHL6 (n=5; 3%); MYD88 (n=5; 3%) and XPO1 (n=5; 3%). At univariate analysis, an adverse impact on time to treatment (TTT) was evident for SF3B1 mutations, higher white blood cell count, higher CLL cells percentage by flow cytometry, CD38 positivity, IGHV unmutated status and at least 2 genomic array abnormalities. Of them, SF3B1 mutations, CLL cells percentage, IGHV unmutated status and number of genomic array aberrations maintained their impact in multivariate analysis. In conclusion, integrating genomic and molecular data, we identified patients at higher risk for treatment need. Therefore, we suggest to evaluate these factors for a better prognostic stratification of normal karyotype CLL subset.
2017-08-31 | GSE103306 | GEO
Project description:About 40% of patients with myelodysplastic syndromes (MDS) present with a normal karyotype and they are facing different courses of disease. To advance the biological understanding and find molecular prognostic markers we performed a high resolution oligonucleotide array study of 107 MDS patients (FAB) with a normal karyotype. Recurrent hidden deletions overlapping with known cytogenetic aberrations or sites of known tumor associated genes were identified in 4q24 (TET2, 2x), 5q31.2 (2x), 7q22.1 (3x) and 21q22.12 (RUNX1, 2x). One patient with a 7q22.1 had an additional 5q31.2 deletion of the AML/MDS region; the smallest deletion identified so far and includes the putative tumor suppressor (ts) genes EGR1 and CTNNA1. One TET2 deletion was homozygous and one heterozygous, with a missense mutation in the remaining allele, further supporting its role as ts gene. Besides these recurrent alterations, additional individual imbalances were found in 34 cases; in total 42/107 (39%) cases had genomic imbalances, deletions were more frequent than gains. These patients had an inferior survival as compared with the rest of the patients (P=0.002). This study emphasizes the heterogeneity of MDS but points to interesting genes which may have diagnostic and prognostic impact. Array CGH experiment, MDS tumor cells vs. control DNA. Total of 107 tumors.
2011-01-30 | E-GEOD-24602 | biostudies-arrayexpress
Project description:Control of oxidative stress in the bone marrow (BM) is key for maintaining the balance between self-renewal, proliferation, and differentiation of hematopoietic cells. Breakdown of this regulation can lead to diseases characterized by BM failure such as the myelodysplastic syndromes (MDS). To better understand the role of oxidative stress in MDS development, we compared protein carbonylation as an oxidative stress marker in BM of patients with MDS and control subjects, and also patients with MDS under treatment with the iron chelator deferasirox.
2019-10-30 | PXD013609 | Pride
Project description:Genomic DNA from sporadic breast tumours was isolated and analysed using array CGH. The NKI 1MB BAC/PAC micro array was used to identify chromosomal aberrations of all tumours. Keywords: sporadic breast tumour, CGH
2010-07-05 | GSE9114 | GEO
Project description:Background & Aims: The metastatic process is complex and remains a major obstacle in the management of colorectal cancer (CRC). To gain a better insight into the biologic events driving the metastatic process we investigated genomic aberrations in a large cohort of matched CRC primaries and distant metastases from various sites. Methods: In total, 62 primary colorectal cancers, 62 matched normal specimens, and 68 matched metastases (from liver, lung, ovary, omentum, and distant lymph nodes) were analyzed by high resolution array comparative genomic hybridization (array CGH) for DNA copy number changes. Findings were validated using a publicly available dataset consisting of 21 primary tumors and matched liver metastases. Fluorescence in situ hybridization (FISH) was used to confirm some of the DNA copy number changes observed. Results: Overall patterns of DNA copy number aberrations were highly similar between primary tumors and their metastases, confirming clonality. Additional copy number aberrations in metastasis are rare and rather than recurrent they were sporadic for individual patients. The only recurrent differences between primary tumors and their metastases were two chromosomal regions, 6q21 and 8q24.21 encompassing the MYC oncogene, that coamplified in three metastases of two patients (3.2%). FISH analysis confirmed the high level co-amplification in the metastasis, which were not detected in their primary tumors. Conclusions: Primary CRC and their metastases show highly similar patterns of DNA copy number changes, additional copy number aberrations in metastasis are rare and recurrences exceptional. These observations are consistent with the hypothesis that the metastatic potential is predestined early in the development of the primary tumor. In total, 62 primary colorectal cancers, 62 matched normal specimens, and 68 matched metastases (liver, lung, ovarian, omentum and distant lymph nodes) were analyzed by high resolution array comparative genomic hybridization (array CGH).
2014-02-10 | E-GEOD-38479 | biostudies-arrayexpress
Project description:Genomic DNA from sporadic breast tumours was isolated and analysed using array CGH. The NKI 1MB BAC/PAC micro array was used to identify chromosomal aberrations of all tumours. Other profiles are located at: GSE9114 Keywords: sporadic breast tumour, CGH.
2010-10-26 | GSE22401 | GEO