Fluorescence in situ hybridization analysis of the ALK gene in 2,045 non-small cell lung cancer patients from North-Western Spain (Galicia).

Identification of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements is a standard diagnostic test in patients with advanced non-small cell lung cancer (NSCLC). The current study describes the experience of ALK rearrangement detection of a referral center in the public health care system of Galicia in North-Western Spain. The fluorescence in situ hybridization (FISH) patterns of the ALK gene and the clinical and pathological features of these patients are reported. This study is also of interest for comparative purposes due to the relative geographical isolation of the area, which could have contributed to particular genetic features. A total of 2,045 tissue samples from NSCLC patients were collected between October 2010 and July 2015 and tested for ALK rearrangements by FISH. Examination of 1,686 paraffin-embedded tissue specimens and 395 cytological samples (306 cell block preparations and 53 cytological smears) was conducted, and any associations between the FISH results and clinicopathological features were assessed. The rate of successful evaluation was marginally higher in tissue samples than in cytological samples (92.9% vs. 84.1%); this difference was not significant. ALK rearrangements were identified in 82 patients(4%): 65 (79.3%) in tissue specimens, 15 (18.3%) in cell block samples and 2 (2.4%) in cytological smears. This genetic translocation appeared to be associated with a non-smoking history, younger age, female gender, stage IV and adenocarcinoma histological type. The findings demonstrate that ALK evaluation by FISH is feasible in tissue and cytological samples. The clinical and pathological features of the ALK-positive series of patients are similar to those previously reported in the literature.

Coronary artery disease is associated with higher epicardial retinol-binding protein 4 (RBP4) and lower glucose transporter (GLUT) 4 levels in epicardial and subcutaneous adipose tissue.

OBJECTIVE: Retinol-binding protein 4 (RBP4), produced by adipocytes and hepatocytes, contributes to an unfavourable lipid profile and insulin resistance, which can contribute to the development of coronary artery disease (CAD). Recently, several studies have shown that epicardial adipose tissue (EAT) differs from subcutaneous adipose tissue (SAT) and plays a role on the physiopathology of CAD because of its proximity to the coronary arteries. We aimed to study the expression and secretion levels of RBP4 in both fat tissues and explore its possible association with CAD. RESEARCH DESIGN AND METHODS: Fifty-eight patients undergoing heart surgery were included in the study. We analysed RBP4 mRNA expression by real-time PCR, protein expression by Western blot and immunohistochemistry, and secretion of EAT and SAT explants from CAD and non-CAD patients by Enzyme Immunoassay. RESULTS: Retinol-binding protein 4 is expressed at similar levels in EAT and SAT, mainly from adipocytes. Protein levels were higher in EAT from CAD than non-CAD patients (0·63 ± 0·09 arbitrary units (a.u).; n = 10) vs (0·41 ± 0·04 a.u.; n = 13, P = 0·039). In contrast, GLUT4 mRNA levels were lower in EAT from CAD than non-CAD patients (6·55 ± 0·16 a.u.; n = 13) vs (7·21 ± 0·18 a.u.; n = 14, P = 0·012). We also found differential expression in SAT between samples from CAD and non-CAD patients [(6·63 ± 0·16 a.u.; n = 14) vs (7·21 ± 0·14 a.u.; n = 14, P = 0·009)]. Besides, EAT releases higher RBP4 levels than SAT after 3, 6, 24 and 48 h of culture. These levels were independent of CAD but significantly higher in diabetic than nondiabetic patients. CONCLUSION: Retinol-binding protein 4 levels behave differently in EAT and SAT with respect to CAD. However, both adipose tissues have lower GLUT4 levels in patients with CAD. These findings suggest a differential regulation of RBP4 production in EAT and SAT that may be influenced by local factors.

Fluorescent in situ hybridization heating pretreatment: the key is temperature control.

Fluorescent in situ hybridization (FISH) is a very useful tool for diagnostic and prognostic purposes in pathology. However, many laboratories still experience troubles when applying FISH to paraffin material. To overcome these difficulties, different pretreatments which include enzymatic digestion have been described. Usually, previous to digestion, a heating step is performed. The aim of this study was to compare the efficiency of the heating step with different buffers and different heating methods. We conclude that the main factor in the heating pretreatment is the temperature control, irrespective of the buffer used. Best results are obtained with any buffer by heating the slides to 99°C for 15 min followed by 10 min at room temperature.

Relationship between epicardial adipose tissue adipocyte size and MCP-1 expression.

Adipocyte size has been associated to increase in inflammatory cytokines expression that can be related to the cardiovascular risk of obesity. Epicardial adipose tissue (EAT) was discovered to play a key role in cardiovascular diseases by producing several inflammatory adipokines. We sought to study whether EAT and subcutaneous adipose tissue (SAT) mean adipocyte sizes are related to the expression of adipokines in patients with cardiovascular diseases. We collected EAT, SAT and blood samples from 22 patients aged 70.9 (s.d. 10.3) undergoing heart surgery. Monocyte chemoattractant protein (MCP)-1, interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha were analyzed by real time RT-PCR, ELISA or immunohistochemistry. Hematoxylin-eosin staining was used for adipocyte area calculations. Adipocyte size is negatively correlated to MCP-1 expression (r=-0.475; p=0.034) in EAT and positively correlated in SAT (r=0.438; p=0.047). These trends persisted after stratification for sex and coronary artery disease (CAD), but only the relationship between EAT MCP-1 and adipocyte size reached statistical significance in the larger group of men with CAD. We have observed that SAT adipocyte size is correlated to BMI (r=0.601; p=0.003); whereas only a non-statistically significant trend was observed in EAT. IL-10 and TNF-alpha expression were not associated to adipocyte size in EAT nor SAT. Secondarily, we found that EAT IL-10 expression is higher in patients with CAD. These results suggest that adipocyte size is a negative determinant of MCP-1 expression in EAT and a positive determinant in SAT. These data might partly explain the different implications of EAT and SAT in cardiovascular diseases.