THU0482 Gene Expression Profiling of Whole Blood Samples of TRAPS Patients Shows Insight into the Molecular Pathogenesis of TRAPS and Response to Canakinumab Treatment

271 Background: CTC RNA analysis currently involves single cell recovery that is laborious and expensive, or alternatively lysis of preserved whole blood which yields RNA predominantly from leukocytes which vastly outnumber CTCs. To effectively characterize gene expression in large patient cohorts, new enrichment methodologies are needed that yield high purity CTC populations while preserving RNA integrity. Here we describe a simple yet robust method for enrichment of prostate CTCs for gene expression analysis. Methods: Blood was drawn with informed consent under an IRB-approved protocol. For initial optimization, CFSE-stained PCa cells were spiked into healthy blood and recovered using various combinations of 2 methods: microfluidic enrichment (Parsortix™ system) and CD45 depletion. For assay qualification, a prostate-specific multiplexed qRT-PCR gene expression panel was developed. Enrichment and gene expression were tested initially using PCa cell lines spiked into healthy blood, then metastatic castrate resistant prostate cancer (mCRPC) blood samples in parallel with CellSearch enumeration. Results: Optimal enrichment of live cells was achieved with CD45 depletion followed by microfluidic enrichment, resulting in an average spiked cell recovery of 30% and approximately 100 contaminating background leukocytes. Using this enrichment method, prostate specific genes were detectable by multiplexed qRT-PCR down to 25 cells spiked into 7.5 ml whole blood, and transcripts were not measurable in matched healthy blood controls. When applied to mCRPC patient blood containing CTCs by CellSearch, multiplexed qRT-PCR successfully detected prostate specific genes in all samples. Conclusions: We developed a novel enrichment method capable of rapidly and efficiently recovering live CTCs with high purity, free of magnetic beads and with very few background leukocytes. Captured cells yielded high-quality RNA with high sensitivity and specificity for prostate-specific transcripts. This approach is applicable to high throughput gene expression profiling assays and offers an alternative to laborious single cell recovery or non-cancer-specific whole blood fixation.

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Gene expression profiling in whole blood of patients with coronary artery disease

Clinical Science ◽

10.1042/cs20100043 ◽

2010 ◽

Vol 119 (8) ◽

pp. 335-343 ◽

Cited By ~ 86

Author(s):

Chiara Taurino ◽

William H. Miller ◽

Martin W. McBride ◽

John D. McClure ◽

Raya Khanin ◽

...

Keyword(s):

Gene Expression ◽

Coronary Artery Disease ◽

Coronary Artery ◽

Gene Expression Profiling ◽

Whole Blood ◽

Expression Profiling ◽

Gene Expression Analysis ◽

Differentially Expressed ◽

Differentially Expressed Mirnas ◽

Artery Disease

Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease.

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