Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples

Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples

Vitamins D2 and D3 Have Overlapping But Different Effects on the Human Immune System Revealed Through Analysis of the Blood Transcriptome

Vitamin D is best known for its role in maintaining bone health and calcium homeostasis. However, it also exerts a broad range of extra-skeletal effects on cellular physiology and on the immune system. Vitamins D(2) and D(3) share a high degree of structural similarity. Functional equivalence in their vitamin D-dependent effects on human physiology is usually assumed but has in fact not been well defined experimentally. In this study we seek to redress the gap in knowledge by undertaking an in-depth examination of changes in the human blood transcriptome following supplementation with physiological doses of vitamin D(2) and D(3). Our work extends a previously published randomized placebo-controlled trial that recruited healthy white European and South Asian women who were given 15 µg of vitamin D(2) or D(3) daily over 12 weeks in wintertime in the UK (Nov-Mar) by additionally determining changes in the blood transcriptome over the intervention period using microarrays. An integrated comparison of the results defines both the effect of vitamin D(3) or D(2) on gene expression, and any influence of ethnic background. An important aspect of this analysis was the focus on the changes in expression from baseline to the 12-week endpoint of treatment within each individual, harnessing the longitudinal design of the study. Whilst overlap in the repertoire of differentially expressed genes was present in the D(2) or D(3)-dependent effects identified, most changes were specific to either one vitamin or the other. The data also pointed to the possibility of ethnic differences in the responses. Notably, following vitamin D(3) supplementation, the majority of changes in gene expression reflected a down-regulation in the activity of genes, many encoding pathways of the innate and adaptive immune systems, potentially shifting the immune system to a more tolerogenic status. Surprisingly, gene expression associated with type I and type II interferon activity, critical to the innate response to bacterial and viral infections, differed following supplementation with either vitamin D(2) or vitamin D(3), with only vitamin D(3) having a stimulatory effect. This study suggests that further investigation of the respective physiological roles of vitamin D(2) and vitamin D(3) is warranted

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples

Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma.

Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours

Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma

Background and purpose: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Materials and methods: Tumour samples (n = 201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/μl). Intra- (CoV = 6.9%) and inter- (CoV = 2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO2. TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p < 0.01) and positive pimonidazole scores (p = 0.005). Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material. © 2012 Elsevier Ltd. All rights reserved