Immunohistochemical localization of epidermal growth factor receptor in leiomyomas from women treated with goserelin depot.

Epidermal growth factor receptors (EGFr) were studied by immunohistochemistry in human myometrium and leiomyomas from women treated with the GnRH agonist (GnRH-a) goserelin. The results were compared with those obtained from women not treated. Vessel cells of leiomyomas and myometrium were always more immunoreactive than fibrocytes and myocites. In fibroids from treated patients the histochemical score (HSCORE) of vessel cells was always significantly lower (p less than 0.001) than in control patients. Our data confirm a role of EGF also in fibroid growth and suggest that, the blood supply reduction associated with the volume reduction of these tumours, consequent to the GnRH-a treatment, could be EGF mediated.     

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.     

Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

Background

Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.

Methodology/Principal Findings

We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.

Conclusions

Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.     

Analysis of diaspore morphology and seed germination in Bubon macedonicum L., a rare species in Italy

Bubon macedonicum L. is a chasmophytic species of south-eastern Europe. In Italy, it has been detected only in Rocca Monforte (Campobasso, central Italy). This rare species is included in the IUCN Red Lists of Critically Endangered Italian Flora, and there are no studies relating to B. macedonicum biology. The seed germination dynamics of this species was studied with the aim of building up an appropriate germination protocol to be used in ex situ conservation. On the basis of an ISTA protocol, about 3,000 seeds were collected from Rocca Monforte in August 2013. Fifty seeds were measured. The considered parameters were seed length, width, thickness, seed surface, volume, density, surface/mass ratio and eccentricity index. The morphometric parameters examined showed morphological dormancy, where a short warm period is necessary for embryo growth and seed germination. The results showed high germination percentages under the different conditions of temperature, pH, GA₃ and photoperiod. Only at 5 °C was there no germination. Finally, the seeds maintain high germination percentages from the seed storage process after 130 and 390 days. This factor can be considered of great importance for the conservation of B. macedonicum over the medium and long term.     

Expression and prognostic significance of the autoimmune regulator gene in breast cancer cells

The autoimmune regulator gene (AIRE) plays a fundamental role in tolerance by promoting the expression of tissue-specific antigens in medullary thymic epithelial cells (mTECs). Recently, AIRE expression was detected also in human keratinocytes and in tumors originating in stratified epithelia. Here, we tested whether AIRE is expressed in cancer cells. We analyzed AIRE expression in cancer cases from The Cancer Genome Atlas (TCGA) RNA-seq dataset and we found association with better outcome. AIRE protein expression was verified by immunohistochemistry in a cohort of 39 human breast cancer specimens and its prognostic relevance was confirmed in microarray-based gene expression data set NKI-295 and KM-Plotter. Both in the RNA-seq and gene expression datasets analyzed, AIRE expression was an independent strong prognostic factor for relapse-free survival (RFS), particularly in estrogen receptor-positive tumors. Enrichment of translation-related pathways was observed in AIRE-expressing tumors by Ingenuity Pathway Analysis and a significant increase of cells in G1 phase and activation of caspase cascades was induced by AIRE transfection in breast cancer luminal cell lines, suggesting that AIRE-induced over-translation of proteins lead to cycle arrest and apoptosis. These data are the first to identify AIRE expression in breast cancer and an association with prognosis.     

Questioning the utility of pooling samples in microarray experiments with cell lines

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.