Project description:Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin fixed paraffin embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumor banks. Once samples are fixed in formalin the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study we have evaluated the whole genome DASL assay from Illumina to perform transcriptomic analysis from archived breast tumor tissue fixed in formalin paraffin embedded blocks. We profiled 76 familial breast tumors from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r2=0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumor molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterisation of many diseases. RNA was extracted from FFPE Familial breast tumours and analysed using the WG-DASL assay for Illumina.
Project description:First line chemotherapy with platinum and cetuximab is usually offered to RM-HNSCC pts. In the Extreme trial a median progression free survival (PFS) time of 5.6 months was reported. However, a small fraction of pts achieves a prolonged PFS (> than 12 months). Till now, no recognized predictive biological factor has been identified. A group of 14 cases treated with a first line platinum and cetuximab with a PFS exceeding 12 months (long PFS) and a group of 26 pts with a PFS less than 5.6 months (short PFS) were selected. The 2 groups were well balanced in regard to recognized prognostic factors (performance status, weight loss, prior radiotherapy, tumor grade, site of primary tumor, residual disease at primary tumor site). Tumor specimens of the recurrence or, if not available, of the primary tumor were collected. In order to identify molecular profiles deregulated between the 2 groups, a gene expression microarray analysis was performed using the Whole-Genome DASL assay and HumanHT-12_v4 BeadArray chips (lllumina). Gender: male=0; female=1. Age: years at diagnosis Site of primary: oral cavity=0; oropharynx=1; hypopharynx=2; larynx=3. Stage of T at first diagnosis according to AJCC 8th edition Grading according to WHO: well differentiated=1; moderately differentiated=2; scarcely differentiated=3. Radiotherapy (RT) prior to recurrence: No=0; Yes=1. Progression Free Survival (PFS): Long (>12 months); Short(<5 months).
Project description:Vitiligo is an acquired depigmentation of the skin inducing a marked alteration of the quality of life of affected individuals. Halting the disease progression and repigmenting the lesional skin represent the two faces of the therapeutic challenge in vitiligo. So far, none of them has been successfully addressed. Oxidative stress and immune system in genetically predisposed individuals participate to the complex pathophysiology of vitiligo. We performed a transcriptome and proteomic analysis on lesional, perilesional and non-depigmented skin of vitiligo patients compared to matched skin controls of healthy subjects. Our results show that the WNT pathway, implicated in melanocytes differentiation, was found to be altered in vitiligo skin. We demonstrated that the oxidative stress decreases WNT expression/activation in keratinocytes and in melanocytes. We developed an ex vivo skin model that remains functional up to 15 days. We then confirmed the decreased activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated the ex vivo depigmented skins from vitiligo patients and successfully induced the differentiation of resident stem cells into pre-melanocytes supporting further exploration of WNT activators to repigment vitiligo lesions. Total of 40 chips. 10 patients (3 biospies per patient: 1 lesional , 1 perilesional and 1 non lesional) ; 10 healthy volunteers (1biopsy in matched anatomical areas)
Project description:Analysis of gene expression changes in tumour epithelium (DCIS and invasive breast cancer) and stroma both immediately surrounding the lesions and more distantly. Total RNA obtained from Formalin Fixed Paraffin Embedded archival material and the individual compartments (stroma and epithelium) compared independently across the samples. Sample abbreviation key: BC = breast cancer DCIS = ductal carcinoma in situ IDC = invasive ductal carcinoma RM = remote metastasis S = stroma NS = near stroma.
Project description:RNA extracted from 78 FFPE tongue samples, 62 tongue carcinomas and 16 non-malignant controls, were succesfully analysed using the whole genome DASL array to obtain gene expression profiles. Gene expression profiles were used to identify differentially expressed genes with the ultimate goal of finding out their importance for tongue cancer development and maintenance. Sample were formalin fixed paraffin embedded biopsies taken for diagnostic purposes and had been stored between 1 and 13 years. Because of the general poor quality of RNA from archival samples a special focus were put on its effect on the detected expression levels. 28 tumours and 16 controls with a CTdiff< 5 were selected for differential gene expression analysis. Raw data for only these samples were normalized again. Data for these 44 samples can be found here. Total RNA from formalin fixed paraffin embedded sample was extracted using the High Pure RNA Paraffin Kit according to the manufacturer’s protocol. Quality of FFPE sample were evaluated by comparing how well a kousekeeping gene (TUBA6) could be amplified using q-PCR in each FFPE sample compared to two fresh frozen sample (Ctdiff= CTffpe-Ctff). The DASL platform (cDNA-mediated annealing, selection, extension, and ligation assay) was used together with the whole genome arrays to obtain expression data for 20 818 genes.
Project description:RNA extracted from 78 FFPE tongue samples, 62 tongue carcinomas and 16 non-malignant controls, were succesfully analysed using the whole genome DASL array to obtain gene expression profiles. Gene expression profiles were used to identify differentially expressed genes with the ultimate goal of finding out their importance for tongue cancer development and maintenance. Sample were formalin fixed paraffin embedded biopsies taken for diagnostic purposes and had been stored between 1 and 13 years. Because of the general poor quality of RNA from archival samples a special focus were put on its effect on the detected expression levels. 28 tumours and 16 controls with a CTdiff< 5 were selected for differential gene expression analysis. Data for only these sample were normalized again and can be found in seperate data file. Total RNA from formalin fixed paraffin embedded sample was extracted using the High Pure RNA Paraffin Kit according to the manufacturer’s protocol. Quality of FFPE sample were evaluated by comparing how well a kousekeeping gene (TUBA6) could be amplified using q-PCR in each FFPE sample compared to two fresh frozen sample (Ctdiff= CTffpe-Ctff). The DASL platform (cDNA-mediated annealing, selection, extension, and ligation assay) was used together with the whole genome arrays to obtain expression data for 20 818 genes.
Project description:This SuperSeries is composed of the following subset Series: GSE34105: Gene expression profiling of archival tongue carcinoma and normal tongue tissue (all samples) GSE34106: Gene expression profiling of archival tongue carcinoma and normal tongue tissue (subset) Refer to individual Series
Project description:Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL-1β, IL-6, IL-8, IL-23p19, TNF-α, CXCL2 and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease. Gut specimens were derived from individuals undergoing resection for localized colon cancer. Microscopically normal colonic mucosa was dissected from the surgical specimen near the resection margin and immediately subjected to the experimental procedures. After extensive washing the mucus layer was removed by dithiothreitol (DTT) treatment. Subsequently, punches of defined surface area were prepared and denuded of epithelial cells by exposure to EDTA. Samples were collected prior to culturing and washing (control, t = 0 h, total mucosa, TM) as well as after loss of the epithelial layer (t = 5 h, mucosa after loss of epithelial layer, LEL-M) and snap frozen in liquid nitrogen. Lamina Propria (LP) was subsequently isolated via Laser Capture Microdissection (LMD) followed by RNA isolation. Microarray analysis of TM-LP (control) and LEL-LP samples was performed using the WG-DASL Human HT_12 V4 Expression BeadChip Assay from Illumina employing a minimum of 200 ng total RNA per sample. Four replicates from individual experiments were measured for each time point.
Project description:Histologic classification of thymomas has significant limitations since complete surgical excision can be curative. In order to better understand the biology of the disease processes, we performed whole genome gene expression analysis. RNA was extracted from fresh frozen tumors from 36 patients with thymomas and follow-up data was available. Gene expression data was correlated with clinicopathological data. Using the Illumina BeadStudio® platform and Human Ref-8 Beadchip, gene expression data was analyzed by Partek®, and Ingenuity Pathways Analysis (IPA). Validation of the chosen genes was performed using quantitative real-time RT-PCR (qRT-PCR). Unsupervised clustering resulted in identification of four clusters of tumors (C1-C4). Using IPA, the top significant biological functions and pathways displayed cell cycle related category and genes in C1 and C2. Carbohydrate metabolism and cellular growth and proliferation were among the most significant for C3 and C4, respectively. On the other hand, cancer and metabolism related functions and pathways were prominent in clinical outcome including metastasis and stage. Our gene expression analysis representing one of the largest series in literature, revealed at least four distinct clusters of thymic tumors. The study identified number of metastasis-associated genes that are potential candidates for therapeutics 41 Samples, 3 Cell Line samples with 1 duplicate (total = 4). 37 Patient samples including 1 duplicate (total = 37).