Whole-genome expression analysis of melanoma tumor biopsies from a population-based cohort.
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ABSTRACT: Analysis of gene expression profiles in metastatic melanoma to validate prognostic signatures. Total RNA was extracted from fresh-frozen melanoma tumors and genome-wide expression profiling was performed using Illlumina Human HT-12V4.0 BeadChip arrays by standard methods. After removal of replicate samples there were 214 unique melanoma samples left.
Project description:Genome wide gene expression profiling of response to synthetic progestin ORG2058 in AB32 cells, a PR positive clone of the MCF-10A cell line, was determined after lentiviral transduction with an expression construct encoding human FOXA1. Cells were treated for 6h or 24h with 10nM ORG2058 or vehicle, 48h after transduction with the FOXA1 construct or empty vector control. Gene expression was measured in total RNA my Illumina whole genome gene expression array. Duplicate monolayer cultures of AB32 cells transduced 48h with pCDH-CMV-FOXA1-EF1-copGFP virus or negative control empty vector pCDH-CMV-mcs-EF1-copGFP virus were treated 6h or 24h with 10nM ORG2058 or 0.1% ethanol vehicle control. Cells were harvested for total RNA isolation and each sample was labelled and hybridized to a single Illumina human whole genome microarray, resulting in three replicate datasets for each of four conditions. Data were analysed separately and combined to give a mean signal intensity for each array probe.
Project description:Time course of response to synthetic progestin ORG2058 in T-47D and ZR-75-1 breast cancer cell lines and in two PR positive clones of the MCF-10A cell line: AB9 and AB32. Transcriptional response to synthetic progestin, 10nM ORG2058, was compared between the four cell lines at three treatment times. T-47D breast cancer cells were treated in triplicate with 10nM ORG2058 or ethanol vehicle and harvested at 2, 6 and 24h after treatment for gene expression profiling
Project description:Patients with febrile malaria were recruited in order to determine Peripheral Blood Mononuclear Cell (PBMC) gene expression during malaria. Blood was harvested from patients during the acute phase of the illness, and then patients were given a curative regimen of antimalarials. Three to four weeks after treatment, patients returned to the malaria clinic and blood was collected again, in order that each patient could serve as his or her own control. PBMC were isolated at the time of blood collection and forzen in RNA extraction buffer. At the end of the study, each patient was arrayed for ~47,000 transcripts, comparing gene expression at the end of therapy to that at the beginning. The goal was to determine which genes were altered as a result of disease at least 2 fold in a statistically significant manner and to assess if the genes involved could be related to Toll-like receptor signaling pathways. Approximately 60 genes involved in inflammation were confirmed by qPCR. Gene expression profiles of blood cells from 14 individuals with malaria are compared to gene expression in blood cells from the same 14 individuals medicated and recovered from malaria symptoms (28 arrays in total).
Project description:This SuperSeries is composed of the following subset Series: GSE22153: Gene Experssion Profiling-Based Identification of Molecular Subtypes in Stage IV Melanoma with Different Clinical Outcome (test set) GSE22154: Gene Experssion Profiling-Based Identification of Molecular Subtypes in Stage IV Melanoma with Different Clinical Outcome (validation set) Refer to individual Series
Project description:Microarray analyses using CD166+ and CD166- pancreatic cancer cell lines from both Panc-1 and SW1990 cells. We carried out microarray analyses using the CD166+ and CD166- separated cells from both Panc-1 and SW1990. The quality of the RNA samples was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). We used a Human HT-12v4 Expression BeadChip (Illumina, San Diego, CA) for these analyses. The data were analyzed using BeadStudio software version 3.2.3 (Illumina).
Project description:Purpose: The incidence of malignant melanoma is increasing worldwide in fair-skinned populations. Melanomas respond poorly to systemic therapy, and metastatic melanomas inevitably become fatal. Although spontaneous regression, likely due to immune defense activation, rarely occurs, we lack a biological rationale and predictive markers in selecting patients for immune therapy. Experimental Design: We performed unsupervised hierarchical clustering of global gene expression data from stage IV melanomas in 57 patients. For further characterization, we used immunohistochemistry of selected markers, genome-wide DNA copy number analysis, genetic and epigenetic analysis of the Q3 CDKN2A locus, and NRAS/BRAF mutation screening. Results: The analysis revealed four distinct subtypes with gene signatures characterized by expression of immune response, pigmentation differentiation, proliferation, or stromal composition genes. Although all subtypes harbored NRAS and BRAF mutations, there was a significant difference between subtypes (P < 0.01), with no BRAF/NRAS wild-type samples in the proliferative subtype. Additionally, the proliferative subtype was characterized by a high frequency of CDKN2A homozygous deletions (P < 0.01). We observed a different prognosis between the subtypes (P = 0.01), with a particularly poor survival for patients harboring tumors of the proliferative subtype compared with the others (P = 0.003). Importantly, the clinical relevance of the subtypes was validated in an independent cohort of 44 stage III and IV melanomas. Moreover, low expression of an a priori defined gene set associated with immune response signaling was significantly associated with poor outcome (P = 0.001). Conclusions: Our data reveal a biologically based taxonomy of malignant melanomas with prognostic effect and support an influence of the antitumoral immune response on outcome. Expression profiles of 20 liver and lymphnode metastases. MM76 and MM23 were made in duplicate. Used as validationset in Jönsson et al. Clin Can Res 2010.
Project description:Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMG-CoA reductase. These results suggest that statins should be re-evaluated as anti-leukemia agents, and illustrate the power of merging physiologically-relevant models with high-throughput screening. Freshly isolated LSCe cells were treated with either 5M-BM-5M BRD7116 or DMSO vehicle control for 6 hours in suspension in IMDM (12440053, Invitrogen), 10% FBS. Total RNA was extracted from the cells, labeled, and hybridized to MouseRef-8 v2.0 Expression BeadChips. Hartwell, K.A. et al., In Press (full citation forthcoming)
Project description:Mammary gland (MG) de novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. We hypothesized that de novo lipogenesis, as reflected by incorporation of 13C carbons from [U-13C]glucose into fatty acids (FAs) and glycerol in triglycerides (TG), will be greater: a. in milk than plasma TG, b. during a high carbohydrate (H-CHO) diet than high fat (H-FAT) diet and c. during feeding than fasting. Healthy lactating women were studied on two isocaloric, isonitrogenous diets. On one occasion subjects received diets containing H-FAT or H-CHO diet for 1 week. Incorporation of 13C from infused [U-13C]glucose into FAs and glycerol was measured using GC/MS methodology and gene expression using RNA isolated from breast milk fat globule (MFG). Incorporation of 13C2 into milk FAs, increased with increased chain length of the FAs from C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was not detected in FAs >C16. During feeding, regardless of diets, enrichment of 13C2 in milk FA and 13C3 in milk glycerol were ~3 and ~7 fold higher compared to plasma FA and glycerol, respectively. Following an overnight fast during H-CHO and H-FAT diets study periods, 25% and 6%, respectively, of medium chain FAs (MCFAs, C6-C12) were derived from glucose but increased to 75% and 25% with feeding. The expression of genes involved in FA or glycerol synthesis pathways was unchanged regardless of diet or fast-fed conditions. Conclusions: The human MG is capable of de novo lipogenesis, of primarily MCFAs and glycerol, which is influenced by the macro-nutrient composition of the maternal diet. On day 7 of consumption of each of the two diets milk samples collected from 7 healthy, lean, exclusively breastfeeding women following an overnight fast and feeding conditions [7 x 2 x 2 = 28 samples] were processed for isotope enrichments and RNA isolation from the milk fat globules. The total RNA was utilized for microarray analyses.
Project description:Purpose: The incidence of malignant melanoma is increasing worldwide in fair-skinned populations. Melanomas respond poorly to systemic therapy, and metastatic melanomas inevitably become fatal. Although spontaneous regression, likely due to immune defense activation, rarely occurs, we lack a biological rationale and predictive markers in selecting patients for immune therapy. Experimental Design: We performed unsupervised hierarchical clustering of global gene expression data from stage IV melanomas in 57 patients. For further characterization, we used immunohistochemistry of selected markers, genome-wide DNA copy number analysis, genetic and epigenetic analysis of the Q3 CDKN2A locus, and NRAS/BRAF mutation screening. Results: The analysis revealed four distinct subtypes with gene signatures characterized by expression of immune response, pigmentation differentiation, proliferation, or stromal composition genes. Although all subtypes harbored NRAS and BRAF mutations, there was a significant difference between subtypes (P < 0.01), with no BRAF/NRAS wild-type samples in the proliferative subtype. Additionally, the proliferative subtype was characterized by a high frequency of CDKN2A homozygous deletions (P < 0.01). We observed a different prognosis between the subtypes (P = 0.01), with a particularly poor survival for patients harboring tumors of the proliferative subtype compared with the others (P = 0.003). Importantly, the clinical relevance of the subtypes was validated in an independent cohort of 44 stage III and IV melanomas. Moreover, low expression of an a priori defined gene set associated with immune response signaling was significantly associated with poor outcome (P = 0.001). Conclusions: Our data reveal a biologically based taxonomy of malignant melanomas with prognostic effect and support an influence of the antitumoral immune response on outcome. Expression profiles of 57 lymphnode and subcutaneous melanoma metastases
Project description:Genomic expression profiles of blood and placenta reveal significant immune-related pathways and categories in Chinese women with Gestational Diabetes Gestational diabetes mellitus (GDM) is a complex metabolic disease which occurs in pregnancy with high prevalence, and its pathogenesis remains elusive. Thus far, there has been no comprehensive gene expression profiling in Chinese women with GDM. In this study, we attempt to define the genes and/or pathways that are involved in GDM with Chinese ethnicity, by the Illumina microarray technique. We found 5,197 and 243 genes with significantly altered expression due to GDM in blood and placenta tissues, respectively. Previously known genes (such as TNF, IL1B, LEP, IFNG, and HLA-G) with altered gene expression in GDM were also validated here. In addition, we identified undescribed genes VAV3, PTPN6, CD48 and IL15, in which expression was related to GDM by microarray and Q-RT-PCR assays. To identify GDM-associated pathways, we used analyses with integrated functional annotation (i.e. KEGG) and extracted two significant pathways, which were Natural killer cell mediated cytotoxicity (hsa04650; FDR = 7.10E-09) in blood and Cytokine-cytokine receptor interaction (hsa04060; FDR = 1.07E-03) in placenta tissues. Immune system process (GO: 0002376) was identified by GO analysis with FDR P-value = 7.01E-60 in blood and FDR P-value = 3.57E-08 in placenta tissues, indicating the importance of immunological and inflammatory categories in GDM. Furthermore, despite differences in the quantity of gene expression, we observed a similar functional distribution between expression of blood and placenta tissues in GDM under the categories of immunity. These newly identified key genes and pathways may provide valuable information for the pathogenesis of GDM and advance disease diagnosis, prevention, medication design, and clinical treatment of the disease. One individual GDM blood tissue sample and one pooled GDM blood tissue sample were compared to one pooled healthy blood tissue sample. One pooled GDM placenta tissue sample was compared to one pooled healthy placenta tissue sample.