Concordance of Immunohistochemistry-Based and Gene Expression-Based Subtyping in Breast Cancer.

Background: Use of immunohistochemistry-based surrogates of molecular breast cancer subtypes is common in research and clinical practice, but information on their comparative validity and prognostic capacity is scarce. Methods: Data from 2 PAM50-subtyped Swedish breast cancer cohorts were used: Stockholm tamoxifen trial-3 with 561 patients diagnosed 1976-1990 and Clinseq with 237 patients diagnosed 2005-2012. We evaluated 3 surrogate classifications; the immunohistochemistry-3 surrogate classifier based on estrogen receptor, progesterone receptor, and HER2 and the St. Gallen and Prolif surrogate classifiers also including Ki-67. Accuracy, kappa, sensitivity, and specificity were computed as compared with PAM50. Alluvial diagrams of misclassification patterns were plotted. Distant recurrence-free survival was assessed using Kaplan-Meier plots, and tamoxifen treatment benefit for luminal subtypes was modeled using flexible parametric survival models. Results: The concordance with PAM50 ranged from poor to moderate (kappa = 0.36-0.57, accuracy = 0.54-0.75), with best performance for the Prolif surrogate classification in both cohorts. Good concordance was only achieved when luminal subgroups were collapsed (kappa = 0.71-0.69, accuracy = 0.90-0.91). The St. Gallen surrogate classification misclassified luminal A into luminal B; the reverse pattern was seen with the others. In distant recurrence-free survival, surrogates were more similar to each other than PAM50. The difference in tamoxifen treatment benefit between luminal A and B for PAM50 was not replicated with any surrogate classifier. Conclusions: All surrogate classifiers had limited ability to distinguish between PAM50 luminal A and B, but patterns of misclassifications differed. PAM50 subtyping appeared to yield larger separation of survival between luminal subtypes than any of the surrogate classifications.

Motor Function Deficits in the Estrogen Receptor Beta Knockout Mouse: Role on Excitatory Neurotransmission and Myelination in the Motor Cortex.

BACKGROUND: Male estrogen receptor beta (ERß) knockout (BERKO) mice display anxiety and aggression linked to, among others, altered serotonergic signaling in the basolateral amygdala and dorsal raphe, impaired cortical radial glia migration, and reduced GABAergic signaling. The effects on primary motor cortex (M1 cortex) and locomotor activity as a consequence of ERß loss have not been investigated. OBJECTIVE: The aim of this study was to determine whether locomotor activity is altered as a consequence of the changes in the M1 cortex. METHODS: The locomotor activity of male wild-type (WT) and BERKO mice was evaluated using the open-field and rotarod tests. Molecular changes in the M1 cortex were analyzed by RNA sequencing, electron microscopy, electrophysiology, and immunohistological techniques. In addition, we established oligodendrocyte (OL) cultures from WT and BERKO mouse embryonic stem cells to evaluate OL function. RESULTS: Locomotor profiling revealed that BERKO mice were more active than WT mice but had impaired motor coordination. Analysis of the M1 cortex pointed out differences in synapse function and myelination. There was a reduction in GABAergic signaling resulting in imbalanced excitatory and inhibitory neurotransmission as well as a defective OL differentiation accompanied by myelin defects. The effects of ERß loss on OL differentiation were confirmed in vitro. CONCLUSION: ERß is an important regulator of GABAergic interneurons and OL differentiation, which impacts on adult M1 cortex function and may be linked to increased locomotor activity and decreased motor coordination in BERKO mice.

MANF protects human pancreatic beta cells against stress-induced cell death.

AIMS/HYPOTHESIS: There is a great need to identify factors that could protect pancreatic beta cells against apoptosis or stimulate their replication and thus prevent or reverse the development of diabetes. One potential candidate is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein. Manf knockout mice used as a model of diabetes develop the condition because of increased apoptosis and reduced proliferation of beta cells, apparently related to ER stress. Given this novel association between MANF and beta cell death, we studied the potential of MANF to protect human beta cells against experimentally induced ER stress. METHODS: Primary human islets were challenged with proinflammatory cytokines, with or without MANF. Cell viability was analysed and global transcriptomic analysis performed. Results were further validated using the human beta cell line EndoC-ßH1. RESULTS: There was increased expression and secretion of MANF in human beta cells in response to cytokines. Addition of recombinant human MANF reduced cytokine-induced cell death by 38% in human islets (p < 0.05). MANF knockdown in EndoC-ßH1 cells led to increased ER stress after cytokine challenge. Mechanistic studies showed that the protective effect of MANF was associated with repression of the NF-κB signalling pathway and amelioration of ER stress. MANF also increased the proliferation of primary human beta cells twofold when TGF-ß signalling was inhibited (p < 0.01). CONCLUSIONS/INTERPRETATION: Our studies show that exogenous MANF protein can provide protection to human beta cells against death induced by inflammatory stress. The antiapoptotic and mitogenic properties of MANF make it a potential therapeutic agent for beta cell protection.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium.

Understanding the normal state of human tissue transcriptome profiles is essential for recognizing tissue disease states and identifying disease markers. Recently, the Human Protein Atlas and the FANTOM5 consortium have each published extensive transcriptome data for human samples using Illumina-sequenced RNA-Seq and Heliscope-sequenced CAGE. Here, we report on the first large-scale complex tissue transcriptome comparison between full-length versus 5'-capped mRNA sequencing data. Overall gene expression correlation was high between the 22 corresponding tissues analyzed (R > 0.8). For genes ubiquitously expressed across all tissues, the two data sets showed high genome-wide correlation (91% agreement), with differences observed for a small number of individual genes indicating the need to update their gene models. Among the identified single-tissue enriched genes, up to 75% showed consensus of 7-fold enrichment in the same tissue in both methods, while another 17% exhibited multiple tissue enrichment and/or high expression variety in the other data set, likely dependent on the cell type proportions included in each tissue sample. Our results show that RNA-Seq and CAGE tissue transcriptome data sets are highly complementary for improving gene model annotations and highlight biological complexities within tissue transcriptomes. Furthermore, integration with image-based protein expression data is highly advantageous for understanding expression specificities for many genes.