Identification of reference genes for quantitative RT-PCR in ascending aortic aneurysms.

Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ≤70 years) with different morphologies of the aortic valve (tricuspid undilated n = 24, dilated n = 11; bicuspid undilated n = 6, dilated n = 15; unicuspid dilated n = 4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue.

Obesity and survival in operable breast cancer patients treated with adjuvant anthracyclines and taxanes according to pathological subtypes: a pooled analysis.

INTRODUCTION Obesity is an unfavorable prognostic factor in breast cancer (BC) patients regardless of menopausal status and treatment received. However, the association between obesity and survival outcome by pathological subtype requires further clarification. METHODS We performed a retrospective analysis including 5,683 operable BC patients enrolled in four randomized clinical trials (GEICAM/9906, GEICAM/9805, GEICAM/2003-02, and BCIRG 001) evaluating anthracyclines and taxanes as adjuvant treatments. Our primary aim was to assess the prognostic effect of body mass index (BMI) on disease recurrence, breast cancer mortality (BCM), and overall mortality (OM). A secondary aim was to detect differences of such prognostic effects by subtype. RESULTS Multivariate survival analyses adjusting for age, tumor size, nodal status, menopausal status, surgery type, histological grade, hormone receptor status, human epidermal growth factor receptor 2 (HER2) status, chemotherapy regimen, and under-treatment showed that obese patients (BMI 30.0 to 34.9) had similar prognoses to that of patients with a BMI < 25 (reference group) in terms of recurrence (Hazard Ratio [HR] = 1.08, 95% Confidence Interval [CI] = 0.90 to 1.30), BCM (HR = 1.02, 0.81 to 1.29), and OM (HR = 0.97, 0.78 to 1.19). Patients with severe obesity (BMI ≥ 35) had a significantly increased risk of recurrence (HR = 1.26, 1.00 to 1.59, P = 0.048), BCM (HR = 1.32, 1.00 to 1.74, P = 0.050), and OM (HR = 1.35, 1.06 to 1.71, P = 0.016) compared to our reference group. The prognostic effect of severe obesity did not vary by subtype. CONCLUSIONS Severely obese patients treated with anthracyclines and taxanes present a worse prognosis regarding recurrence, BCM, and OM than patients with BMI < 25. The magnitude of the harmful effect of BMI on survival-related outcomes was similar across subtypes.

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta.

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.

Evaluation of two HbA1c point-of-care analyzers.

BACKGROUND Measurement of HbA1c is the most important parameter to assess glycemic control in diabetic patients. Different point-of-care devices for HbA1c are available. The aim of this study was to evaluate two point-of-care testing (POCT) analyzers (DCA Vantage from Siemens and Afinion from Axis-Shield). We studied the bias and precision as well as interference from carbamylated hemoglobin. METHODS Bias of the POCT analyzers was obtained by measuring 53 blood samples from diabetic patients with a wide range of HbA1c, 4%-14% (20-130 mmol/mol), and comparing the results with those obtained by the laboratory method: HPLC HA 8160 Menarini. Precision was performed by 20 successive determinations of two samples with low 4.2% (22 mmol/mol) and high 9.5% (80 mmol/mol) HbA1c values. The possible interference from carbamylated hemoglobin was studied using 25 samples from patients with chronic renal failure. RESULTS The means of the differences between measurements performed by each POCT analyzer and the laboratory method (95% confidence interval) were: 0.28% (p<0.005) (0.10-0.44) for DCA and 0.27% (p<0.001) (0.19-0.35) for Afinion. Correlation coefficients were: r=0.973 for DCA, and r=0.991 for Afinion. The mean bias observed by using samples from chronic renal failure patients were 0.2 (range -0.4, 0.4) for DCA and 0.2 (-0.2, 0.5) for Afinion. Imprecision results were: CV=3.1% (high HbA1c) and 2.97% (low HbA1c) for DCA, CV=1.95% (high HbA1c) and 2.66% (low HbA1c) for Afinion. CONCLUSIONS Both POCT analyzers for HbA1c show good correlation with the laboratory method and acceptable precision.