Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue.

BACKGROUND: There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material. METHODS: Accuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel™ microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model. RESULTS: The overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9-98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and - 0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50 ng and 3.5 µg respectively was conservative. CONCLUSION: The Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.

Lipoprotein subfraction oxidation in acute exercise and ageing.

Exercise and ageing can independently increase free radical production that may enhance the susceptibility of LDL to oxidation and create a more atherogenic LDL particle. This investigation was designed to examine exercise and ageing on the susceptibility of LDL subfractions to oxidation. Eleven aged (55 ± 4 years) and twelve young (21 ± 2 years) participants completed a progressive exercise test to exhaustion and within one week performed a 1 h bout of moderate intensity (65% VO(2max)) exercise. Blood was assayed for metabolites associated with lipid composition (total cholesterol, free cholesterol, triglycerides) and lipoprotein susceptibility to oxidation. Exercise increased small density (sdLDL) oxidation, independently of age (p < 0.05). However, sdLDL oxidation further increased 24 h post exercise in the aged group (p < 0.05). With regards to the changes in lipid components within LDL, free and total cholesterol and triglycerides in large buoyant (lbLDL) were all elevated 24 h post exercise in aged compared with young (p < 0.05 for all comparisons). There was a decrease in triglycerides in medium density (mdLDL) 24 h post exercise in the aged group (p < 0.05). The lipid composition of sdLDL, VLDL, HDL(2), HDL(3) and serum lipid hydroperoxides remained unchanged as a function of exercise and ageing (p > 0.05). Although regular exercise training is known to be protective against cardiovascular disease (CVD) onset, our data demonstrates that acute exercise can increase sdLDL oxidative susceptibility, and this is independent of age and regardless of a change in LDL lipid composition. However, age seems to be a determining factor with regards the susceptibility of sdLDL to oxidation 24 h following exercise.

Cross-laboratory validation of the OncoScan® FFPE Assay, a multiplex tool for whole genome tumour profiling.

BACKGROUND: Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. METHODS: In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). RESULTS: Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. CONCLUSIONS: Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.

Exercise training protects the LDL I subfraction from oxidation susceptibility in an aged human population.

BACKGROUND: Exercise training is considered an effective strategy to improve metabolic disease. Despite this, less is known regarding exercise training in the prevention and susceptibility of LDL subfraction oxidation, particularly in an aged population. METHODS: Eleven aged (55 ± 4 yrs) and twelve young (21 ± 2 yrs) participants were randomly separated into an experimental or control group as follows: young exercise (n = 6); young control (n = 6); aged exercise (n = 6) and aged control (n = 5). The participants assigned to the exercise groups performed 12 weeks of moderate intensity (55-65% VO2max) exercise training. Venous blood was extracted at baseline, and 48 h following 12 weeks of exercise and assayed for a range of metabolites associated with lipid composition and lipoprotein susceptibility to oxidation. RESULTS: Although there was no difference in the oxidation potential (time ½ max) of LDL I, II or III between groups at baseline (p > 0.05), there was an increase in time ½ max for LDL I following exercise within the aged exercise group (p < 0.05). Moreover, α-tocopherol concentration was selectively lower in the aged exercise group, compared to the young exercise at baseline. The lipid composition of LDL I, LDL II, LDL III, VLDL, HDL2, HDL3 and serum lipid hydroperoxides remained unchanged as a function of exercise training and ageing (p > 0.05). CONCLUSION: The primary finding of this study demonstrates that adaptations in LDL resistance to oxidation occur following 12 weeks of exercise training in the aged, and this may be of clinical significance, as oxidation of LDL has been implicated in atherosclerosis.

The effects of aerobic exercise training at two different intensities in obesity and type 2 diabetes: implications for oxidative stress, low-grade inflammation and nitric oxide production.

AIMS: To investigate the effect of 16 weeks of aerobic training performed at two different intensities on nitric oxide (tNOx) availability and iNOS/nNOS expression, oxidative stress (OS) and inflammation in obese humans with or without type 2 diabetes mellitus (T2DM). METHODS: Twenty-five sedentary, obese (BMI > 30 kg/m2) males (52.8 ± 7.2 years); 12 controls versus 13 T2DM were randomly allocated to four groups that exercised for 30 min, three times per week either at low (Fat-Max; 30-40% VO(2max)) or moderate (T(vent); 55-65 % VO(2max)) intensity. Before and after training, blood and muscle samples (v. lateralis) were collected. RESULTS: Baseline erythrocyte glutathione was lower (21.8 ± 2.8 vs. 32.7 ± 4.4 nmol/ml) and plasma protein oxidative damage and IL-6 were higher in T2DM (141.7 ± 52.1 vs. 75.5 ± 41.6 nmol/ml). Plasma catalase increased in T2DM after T(vent) training (from 0.98 ± 0.22 to 1.96 ± 0.3 nmol/min/ml). T2DM groups demonstrated evidence of oxidative damage in response to training (elevated protein carbonyls). Baseline serum tNOx were higher in controls than T2DM (18.68 ± 2.78 vs. 12.34 ± 3.56 µmol/l). Training at T(vent) increased muscle nNOS and tNOx in the control group only. Pre-training muscle nNOS was higher in controls than in T2DMs, while the opposite was found for iNOS. No differences were found after training for plasma inflammatory markers. CONCLUSION: Exercise training did not change body composition or aerobic fitness, but improved OS markers, especially when performed at T(vent). Non-diabetics responded to T(vent) training by increasing muscle nNOS expression and tNOx levels in skeletal muscle while these parameters did not change in T2DM, perhaps due to higher insulin resistance (unchanged after intervention).

Effect of cinnamon on gastric emptying, arterial stiffness, postprandial lipemia, glycemia, and appetite responses to high-fat breakfast.

BACKGROUND: Cinnamon has been shown to delay gastric emptying of a high-carbohydrate meal and reduce postprandial glycemia in healthy adults. However, it is dietary fat which is implicated in the etiology and is associated with obesity, type 2 diabetes and cardiovascular disease. We aimed to determine the effect of 3 g cinnamon (Cinnamomum zeylanicum) on GE, postprandial lipemic and glycemic responses, oxidative stress, arterial stiffness, as well as appetite sensations and subsequent food intake following a high-fat meal. METHODS: A single-blind randomized crossover study assessed nine healthy, young subjects. GE rate of a high-fat meal supplemented with 3 g cinnamon or placebo was determined using the 13C octanoic acid breath test. Breath, blood samples and subjective appetite ratings were collected in the fasted and during the 360 min postprandial period, followed by an ad libitum buffet meal. Gastric emptying and 1-day fatty acid intake relationships were also examined. RESULTS: Cinnamon did not change gastric emptying parameters, postprandial triacylglycerol or glucose concentrations, oxidative stress, arterial function or appetite (p < 0.05). Strong relationships were evident (p < 0.05) between GE Thalf and 1-day palmitoleic acid (r = -0.78), eiconsenoic acid (r = -0.84) and total omega-3 intake (r = -0.72). The ingestion of 3 g cinnamon had no effect on GE, arterial stiffness and oxidative stress following a HF meal. CONCLUSIONS: 3 g cinnamon did not alter the postprandial response to a high-fat test meal. We find no evidence to support the use of 3 g cinnamon supplementation for the prevention or treatment of metabolic disease. Dietary fatty acid intake requires consideration in future gastrointestinal studies. TRIAL REGISTRATION NUMBER: at http://www.clinicaltrial.gov: NCT01350284.

Yap is a novel regulator of C2C12 myogenesis.

The expression, regulation and function of mammalian Hippo pathway members in skeletal muscle is largely unknown. The aim of this study was thus to test the hypothesis that core members of the mammalian Hippo pathway are expressed in skeletal muscle and that the transcriptional co-factor Yap, a core member of the Hippo pathway, regulates C2C12 myogenesis. We found that the major components of the mammalian Hippo pathway including Yap are all expressed in skeletal muscles, C2C12 myoblasts and myotubes. In C2C12 myoblasts, Yap Ser127 phosphorylation is low and Yap localises to nuclei. Upon differentiation, Yap Ser127 phosphorylation increases approximately 20-fold and Yap translocates from the nucleus to the cytosol. To test whether the observed increase of Yap Ser127 phosphorylation is required for differentiation we overexpressed hYAP1 S127A, a mutant that can not be phosphorylated at Ser127, in C2C12 myoblasts. We found that overexpression of hYAP S127A prevented myotube formation, whereas the overexpression of wildtype hYAP1 or empty vector had no effect. In addition, more hYAP1 S127A overexpressing cells progressed through the S phase of the cell cycle and the expression of MRFs (myogenin, Myf5), Mef2c and cell cycle regulators (p21, cyclin D1) was significantly changed when compared to wildtype hYAP1 and empty vector overexpressing cells. This data suggests that the phosphorylation of Yap at Ser127 leads to a changed expression of MRFs and cell cycle regulators and is required for C2C12 myoblasts to differentiate into myotubes.