Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

<p>Abstract</p> <p>Background</p> <p>Real-time quantitative PCR (RQ-PCR) forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC) genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (<it>ESR1</it>) was used a target gene to compare the effect of choice of EC on the estimate of gene quantity.</p> <p>Results</p> <p>The expression and validity of candidate ECs (<it>GAPDH</it>, <it>TFRC</it>, <it>ABL</it>, <it>PPIA</it>, <it>HPRT1</it>, <it>RPLP0</it>, <it>B2M</it>, <it>GUSB</it>, <it>MRPL19</it>, <it>PUM1 </it>and <it>PSMC4) </it>was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. <it>MRPL19 </it>and <it>PPIA </it>were identified as the most stable and reliable EC genes, while <it>GUSB</it>, <it>RPLP0 </it>and <it>ABL </it>were least stable. There was a highly significant difference in variance between ECs. <it>ESR1 </it>expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of <it>ESR1</it>.</p> <p>Conclusion</p> <p>We have validated two endogenous control genes, <it>MRPL19</it> and <it>PPIA</it>, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the quantitation of gene expression data, facilitating the detection of smaller changes in gene expression than otherwise possible. The combination identified here is a good candidate for use as a two-gene endogenous control in a broad spectrum of future research and diagnostic applications in breast cancer.</p

Health outcomes of a subsidised fruit and vegetable program for Aboriginal children in northern New South Wales

Objective: To evaluate the impact of a fruit and vegetable subsidy program on short-term health outcomes of disadvantaged Aboriginal children.Design, setting and participants: A before-and-after study involving clinical assessments, health record audits and blood testing of all children aged 0&ndash;17 years (n = 167) from 55 participating families at baseline and after 12 months at three Aboriginal community-controlled health services in New South Wales. All assessments were completed between December 2008 and September 2010.Intervention: A weekly box of subsidised fruit and vegetables linked to preventive health services and nutrition promotion at an Aboriginal Medical Service.Main outcome measures: Change in episodes of illness, health service and emergency department attendances, antibiotic prescriptions and anthropometry.Results: There was a significant decrease in oral antibiotics prescribed (&minus; 0.5 prescriptions/year; 95% CI, &minus; 0.8 to &minus; 0.2) during 12 months of participation in the program compared with the 12 months before the program. The proportion of children classified as overweight or obese at baseline was 28.3% (38/134) and the proportion in each weight category did not change (P = 0.721) after 12 months. A small but significant increase in mean haemoglobin level (3.1 g/L; 95% CI, 1.4&ndash;4.8 g/L) was shown, although the proportion with iron deficiency (baseline, 41%; follow-up, 37%; P = 0.440) and anaemia (baseline, 8%; follow-up, 5%; P = 0.453) did not change significantly.Conclusion: This fruit and vegetable subsidy program was associated with improvements in some indicators of short-term health status among disadvantaged Aboriginal children. A controlled trial is warranted to investigate the sustainability and feasibility of healthy food subsidy programs in Australia

Circulating microRNAs miR-331 and miR-195 differentiate local luminal a from metastatic breast cancer

BACKGROUND: Breast cancer is the leading cause of cancer related death in women, with metastasis the principle cause of mortality. New non-invasive prognostic markers are needed for the early detection of metastasis, facilitating treatment decision optimisation. MicroRNA (miRNA) are small, non-coding RNAs regulating gene expression and involved in many cellular processes, including metastasis. As biomarkers, circulating miRNAs (in blood) hold great promise for informing diagnosis or monitoring treatment responses. METHODS: Plasma extracted RNA from age matched local Luminal A (n = 4) or metastatic disease (n = 4) were profiled using Next Generation Sequencing. Selected differentially expressed miRNA were validated on a whole blood extracted miRNA cohort [distant metastatic disease (n = 22), local disease (n = 31), healthy controls (n = 21)]. Area Under the Curve (AUC) in Receiver Operating Characteristic (ROC) analyses was performed. RESULTS: Of 4 miRNA targets tested (miR-181a, miR-329, miR-331, miR-195), mir-331 was significantly over-expressed in patients with metastatic disease, compared to patients with local disease (p \u3c 0.001) or healthy controls (p \u3c 0.001). miR-195 was significantly under-expressed in patients with metastatic disease, compared to patients with local disease (p \u3c 0.001) or healthy controls (p = 0.043). In combination, miR-331 and miR-195 produced an AUC of 0.902, distinguishing metastatic from local breast cancer. CONCLUSIONS: We identified and validated two circulating miRNAs differentiating local Luminal A breast cancers from metastatic breast cancers. Further investigation will reveal the molecular role of these miRNAs in metastasis, and determine if they are subtype specific. This work demonstrates the ability of circulating miRNA to identify metastatic disease, and potentially inform diagnosis or treatment effectiveness

Abdominal obesity and other risk factors largely explain the high CRP in Indigenous Australians relative to the general population, but not gender differences: a cross-sectional study

Background: Previous studies reported high C-reactive protein (CRP) levels in Indigenous Australians, which may contribute to their high risk of cardiovascular disease. We compared CRP levels in Indigenous Australians and the general population, accounting for obesity and other risk factors.Methods: Cross-sectional study of CRP and risk factors (weight, height, waist and hip circumferences, blood pressure, lipids, blood glucose, and smoking status) in population-based samples from the Diabetes and Related conditions in Urban Indigenous people in the Darwin region (DRUID) study, and the Australian Diabetes, Obesity and Lifestyle study (AusDiab) follow-up.Results: CRP concentrations were higher in women than men and in DRUID than AusDiab. After multivariate adjustment, including waist circumference, the odds of high CRP (&gt;3.0 mg/L) in DRUID relative to AusDiab were no longer statistically significant, but elevated CRP was still more likely in women than men. After adjusting for BMI (instead of waist circumference) the odds for elevated CRP in DRUID participants were still higher relative to AusDiab participants among women, but not men. Lower HDL cholesterol, impaired glucose tolerance (IGT), and higher diastolic blood pressure were associated with having a high CRP in both men and women, while current smoking was associated with high CRP in men but not women.Conclusions: High concentrations of CRP in Indigenous participants were largely explained by other risk factors, in particular abdominal obesity. Irrespective of its independence as a risk factor, or its aetiological association with coronary heart disease (CHD), the high CRP levels in urban Indigenous women are likely to reflect increased vascular and metabolic risk. The significance of elevated CRP in Indigenous Australians should be investigated in future longitudinal studies

The paradox of odd-even price in fashion luxury sector : empirical evidence from an international direct observation of luxury stores

Price has always had a key role in the luxury fashion market, because high prices are linked to the uniqueness and the prestige of luxury products and brands. Because of this direct contribution of price to the luxury essence, scholars have partially neglected the possible existence of unintuitive and controversial pricing strategies followed by luxury firms. This article deals with this literature gap, particularly analyzing a specific pricing strategy that seems to be in contrast with the nature of luxury pricing: the odd-even price (OEP). With the direct observation of physical and digital store windows of 20 luxury brands, this research investigates the role of OEP in the fashion luxury sector. Particularly, this work examines the relationship between the OEP strategy and the luxury level of fashion brands considering both offline and online channels

Maternal effects on anogenital distance in a wild marmot population

Peer reviewedPublisher PD

Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: stimulation of epithelial to mesenchymal transition (EMT)

Bone marrow-derived mesenchymal stem cells (MSCs) are known to specifically migrate to and engraft at tumour sites. Understanding interactions between cancer cells and MSCs has become fundamental to determining whether MSC-tumour interactions should be harnessed for delivery of therapeutic agents or considered a target for intervention. Breast Cancer Cell lines (MDA-MB-231, T47D & SK-Br3) were cultured alone or on a monolayer of MSCs, and retrieved using epithelial specific magnetic beads. Alterations in expression of 90 genes associated with breast tumourigenicity were analysed using low-density array. Expression of markers of epithelial–mesenchymal transition (EMT) and array results were validated using RQ-PCR. Co-cultured cells were analysed for changes in protein expression, growth pattern and morphology. Gene expression and proliferation assays were also performed on indirect co-cultures. Following direct co-culture with MSCs, breast cancer cells expressed elevated levels of oncogenes (NCOA4, FOS), proto-oncogenes (FYN, JUN), genes associated with invasion (MMP11), angiogenesis (VEGF) and anti-apoptosis (IGF1R, BCL2). However, universal downregulation of genes associated with proliferation was observed (Ki67, MYBL2), and reflected in reduced ATP production in response to MSC-secreted factors. Significant upregulation of EMT specific markers (N-cadherin, Vimentin, Twist and Snail) was also observed following co-culture with MSCs, with a reciprocal downregulation in E-cadherin protein expression. These changes were predominantly cell contact mediated and appeared to be MSC specific. Breast cancer cell morphology and growth pattern also altered in response to MSCs. MSCs may promote breast cancer metastasis through facilitation of EMT.National Breast Cancer Research Institute (NBCRI), a Royal College of Surgeons in Ireland (RCSI) Surgical Research Grant a Health Research Board Project Grant and a ScienceFoundation Ireland CSET award.Deposited by bulk impor

Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against &gt;100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology

Strategic implications of valuation methods

Author's OriginalStrategy is ultimately aimed at creating shareholder value, placing valuation in a central role linking finance and strategy. Focusing on growth options, this paper uses a unique "perfect information" model to examine, from a strategy point of view, the relationship between the market value of the firm and its intrinsic, or DCF, value. Although the research is at the level of the firm, the results have implications at the level of individual strategies and projects, since a firm can be conceptualized as a collection of projects. The findings highlight the relationship between the value of growth options and macroeconomic conditions, industry characteristics, and firm-specific factors. A revised version of this paper has since been published in the journal Advances in Strategic Management. Please use this version in your citations.Alessandri, T. M., Lander, D. M., & Bettis, R. A. (2007), Strategic Implications of Valuation: Evidence from Valuing Growth Options, in Professor Brian Silverman (ed.) Real Options Theory. Advances in Strategic Management, 24, 459-48