Expression of small breast epithelial mucin (SBEM) protein in tissue microarrays (TMAs) of primary invasive breast cancers.

AIMS: Small breast epithelial mucin (SBEM) is a recently described gene product that shows promise as a new breast biomarker. The aim was to investigate for the first time SBEM protein expression in a large cohort (n = 300) of invasive breast cancers, its relationship to established clinical variables and its association with clinical outcome. METHODS AND RESULTS: Immunohistochemical analysis was performed on tissue microarrays consisting of 149 oestrogen receptor (ER) alpha- and 151 ERalpha+ breast cancers. Overall, 18% of tumours were SBEM+ (n = 53/300). However, SBEM protein was more frequently observed in ER- (22%) than in ER+ cancers (13%; P = 0.049). A significant association with psoriasin/S100A7 expression (P < or = 0.0001) was observed in the entire cohort. SBEM was also positively associated with HER-2 (P = 0.046) in ER- cancers, and increased levels of SBEM were strongly associated with higher tumour grade (P = 0.0015). Furthermore, SBEM expression showed a trend towards an association with reduced overall survival and relapse-free survival in the ER+ cohort (P = 0.063 and P = 0.072, respectively). CONCLUSIONS: Our results suggest that SBEM may identify a unique subset of breast cancers with poor prognosis and may have future implications for therapeutic management of this disease.

Potential role of estrogen receptor alpha (ERalpha) phosphorylated at Serine118 in human breast cancer in vivo.

Post-translational modifications of proteins are known to be important in protein activity and ERalpha is known to be phosphorylated at multiple sites within the protein. The exact function of site-specific phosphorylation in ERalpha is unknown, although several hypotheses have been developed using site-directed mutagenesis and cell culture models. Targeting the ERalpha at the level of such post-translational modification pathways would be a new and exciting approach to endocrine therapy in breast cancer, but adequate knowledge is lacking with regard to the relevance of site-specific phosphorylation in ERalpha in human breast cancer in vivo. Recently, antibodies to P-Serine(118)-ERalpha and P-Serine(167)-ERalpha, two major sites of phosphorylation in ERalpha, have become available and some in vivo data are now available to complement studies in cells in culture. However, the in vivo data are somewhat contradictory and limited by the small cohorts used and the lack of standard well-characterized reagents and protocols.

Expression of oestrogen receptor-beta in oestrogen receptor-alpha negative human breast tumours.

To analyse the phenotype of breast tumours that express oestrogen receptor-beta (ERbeta) alone tissue microarrays were used to investigate if ERbeta isoforms are associated with specific prognostic markers and gene expression phenotypes in ERalpha-negative tumours. ERalpha-negative tumours were positive for ERbeta1 in 58% of cases (n=122/210), total ERbeta in 60% (n=115/192) and ERbeta2/cx in 57% of cases (n=114/199). Oestrogen receptor-beta1 and total ERbeta were significantly correlated with Ki67 (r=0.28, P<0.0001, n=209; r=0.29, P<0.0001, n=191) and with CK5/6, a marker of the basal phenotype (r=0.20, P=0.0106, n=170; r=0.18, P=0.0223, n=158). ERbeta2/cx was strongly associated with p-c-Jun and NF-kappaBp65 (r=0.53, P<0.0001, n=93; r=0.35, P<0.0001, n=176). This study shows that a range of ERbeta isoform expression occurs in ERalpha-negative breast tumours. While expression of ERbeta1, total and ERbeta2/cx are correlated, individual forms show associations with certain phenotypes that suggest different roles in subsets of ERalpha-negative cancers. Based on our in vivo observations, ERbeta may have the potential to become a therapeutic target in the specific subcohort of ERalpha-negative breast cancers.

Distinct expression patterns of ER alpha and ER beta in normal human mammary gland.

AIM: Two oestrogen receptors (ERs) have been identified to date-the "classic" ER alpha and the more recently described ER beta. Although much is known about ER alpha at the mRNA and protein levels, our knowledge of the expression and distribution of ER beta protein is much more limited. The aim of this study was to compare the cellular distribution of ER alpha and ER beta in normal human mammary gland. METHODS: Formalin fixed, paraffin wax embedded material was obtained from reduction mammoplasty specimens, normal tissue adjacent to breast tumour, or fibroadenoma. Sections were immunohistochemically stained for ER alpha, ER beta, and the progesterone receptor. The staining pattern for each antibody was evaluated and compared. RESULTS: ER alpha was restricted to the cell nuclei of epithelial cells lining ducts and lobules. Although ER beta was also seen in these cells, additional strong staining was detected specifically in the cell nuclei of myoepithelial cells. Occasional staining was seen in surrounding stromal and endothelial cell nuclei and in lymphocytes. CONCLUSIONS: ER subtypes have distinct distribution patterns in the normal mammary gland. The widespread distribution of ER beta suggests that it may be the dominant ER in the mammary gland where it may be acting as a natural suppressor.

Evaluation of seven oestrogen receptor beta antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast tissue.

Two oestrogen receptors, ER alpha and ER beta, exist. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. The aim of this study was to determine the utility of seven ER beta antibodies (14C8, 8D5, PAI313, PPG5/10, N19, 9.88, and D7N) raised against different domains of ER beta in three commonly used laboratory applications, namely immunohistochemistry, western blot, and flow cytometry, using human breast material. For immunohistochemical analysis of frozen material, PAI313 and D7N gave stronger and more specific signals than 14C8, 8D5, and PPG5/10. In paraffin sections, 14C8, closely followed by PPG5/10, gave by far the most superior nuclear immunoreactivity, compared with the other antibodies tested. In general, flow cytometry results mirrored the immunohistochemistry data for paraffin sections, with antibodies ranked 14C8 > 8D5> or = PAI-313 > PPG5/10 >D7N. For western blotting, 8D5 and D7N yielded the strongest and most consistent bands, with weaker bands seen with the others. It is concluded that ER beta protein can be detected using specific antibodies. However, there is considerable variation between the specificity and application of these antibodies, highlighting the fact that careful optimization is required when selecting an antibody for use in a particular laboratory technique.

Immunohistochemical detection of ERbeta in breast cancer: towards more detailed receptor profiling?

Oestrogen receptor (ER) is used routinely to predict endocrine responsiveness in patients with breast cancer. A second ER, ERbeta has been described but its significance remains undefined; most studies have described mRNA levels rather than protein expression. Here, we demonstrate for the first time, immunohistochemical detection of ERbeta in archival breast tumours.

Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia.

Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.

Induction of nodavirus disease in seabass, Dicentrarchus labrax, using different infection models.

The aim of this study was to investigate the susceptibility of juvenile and adult seabass, which are generally thought to be refractory to nodavirus. Moreover, preliminary immunological studies were performed to examine the immune response of adult seabass. Successful transmission of the disease was experimentally demonstrated in juvenile and adult seabass as ascertained by the presence of the clinical signs of the disease, re-isolation of the virus in the SSN-1 cell line and subsequent confirmation by histopathology and immunohistochemistry. Bigger seabass not only developed the clinical disease but also suffered mortalities. Serum neutralisation titres were considered low in this study.