Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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In vivo and in vitro study of asthma

ABSTRACT: To further development of our gene expression approach to biodosimetry, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish asthma and normal subjects. We analyzed gene expression patterns of the peripheral blood cells from asthma patients compared with those from normal subjects using microarray analyses. In addition, we analyzed gene expression patterns of the LPS or HDM-treated THP1 cells compared with those from non-treated THP1 cells. And we analyzed the microarray data through clustering analysis, signaling pathway analysis and others. Ten milliliters of peripheral blood of five mild asthma, five severe asthma and five normal subjects were centrifuged at 4000 rpm for 20 min to collect 0.4 mL of the buffy coat fraction for in vivo study. To induce an inflammatory response, for in vitro study, the THP1 cells were seeded in 12-well plates at a density of 1 M-CM-^W 106 cells/well in RPMI 1640 medium containing 0.5% fetal bovine serum and stimulated for 24 hrs with LPS or HDM extract. After stimulation the THP1 cells were harvested using a cell scraper and lysed for total RNA extraction. Total RNA was extracted from the buffy coat fractions and RNA was reverse transcribed into cDNA for microarray analysis

ORGANISM(S): Homo sapiens

SUBMITTER: Yoosik Yoon

PROVIDER: E-GEOD-27876 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high intra-tumoral heterogeneity, frequently resistant to treatment and no known targeted therapy available to improve patient outcomes. It has been hypothesized that the genomic architecture of a TNBC tumour evolves over time, both before, and during therapy, leading to therapy resistance and a high propensity to relapse. Whether this is an inherent property of the tumour or acquired over time is not well characterized. Despite this important clinical implication, limited studies have been carried out to unravel temporal evolution of TNBC over time. Herein, we report an OMICS based analysis of three TNBC patients who were longitudinally sampled during their treatment at different times of relapse. We recruited three TNBC patients at the time of their first relapse who were then followed-up through the course of their treatment. We obtained retrospective samples (tumour samples) from patient tumours at diagnosis (before neo-adjuvant chemotherapy - NACT) at surgery (post NACT) and prospectively sampled them at each subsequent relapse (tumour, blood plasma, and buffy coat) as determined by RECIST criteria. Tumor and buffy coat DNA were subjected to whole exome sequencing (WES) at 200x, and SNP arrays for copy number variation (CNV) analysis. RNA from tumour samples at relapse was subjected to whole transcriptome sequencing. Pathogenic germline BRCA1 variants identified in WES were validated using Sanger sequencing. 1084 somatic mutations identified in whole exome sequencing of all tumour tissues (n=13) from three patients, were subjected to a custom amplicon ultra-deep sequencing assay at 30,000X in their germline DNA (n=3), tumour DNA (n=10), and cfDNA from plasma samples at relapse (n=8). Copy number corrected allele frequencies, tumour ploidy, tumour purity, and ultra-deep sequencing assay derived variant allele frequencies were used to infer clonal and phylogenetic architecture of each patient as it evolved under selective pressure of therapy over time. Clonality analysis incorporating allele fractions from ultra-deep sequencing identified clones comprising of mutations that are present throughout the course of therapy which we term as founding clones and stem mutations respectively. Such founding clones comprising of stem mutations in all 3 patients were present throughout the course of treatment, irrespective of change in treatment modalities. These stem clones included well characterized cancer related genes like PDGFRB & ARID2 (Patient 02), TP53, BRAF & CSF3R (Patient 04) and ESR1, APC, EZH2 & TP53 (Patient 07). Such branching evolution is seen in all three patients wherein the dominant clone (stem clone) acquires additional mutations to form sub-clones, while persisting over time. These sub-clones may be chemo and radio resistant, while also providing for organ specific metastatic potential. Allele fractions of expressed variants inferred from RNA-Seq data co-related with allele fractions from WES data indicating that all somatic.

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In vivo and in vitro study of asthma

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