An international reproducibility study validating quantitative determination of ERBB2, ESR1, PGR, and MKI67 mRNA in breast cancer using MammaTyper (R)

Background: Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper (R) test. Methods: Ten international pathology institutions participated in this study and determined messenger RNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper (R) test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss' kappa statistics, and interclass correlation coefficients (ICCs). Results: Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980-0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00. Conclusions: On the basis of these data, the MammaTyper (R) has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.Peer reviewe

Doll Play narratives about starting school in children of socially anxious mothers, and their relation to subsequent child school-based anxiety

Background: Child social anxiety is common, and predicts later emotional and academic impairment. Offspring of socially anxious mothers are at increased risk. It is important to establish whether individual vulnerability to disorder can be identified in young children. Method: The responses of 4.5 year-old children of mothers with social phobia (N = 62) and non-anxious mothers (N = 60) were compared, two months before school entry, using a Doll Play (DP) procedure focused on the social challenge of starting school. DP responses were examined in relation to teacher reports of anxious-depressed symptoms and social worries at the end of the child’s first school term. The role of earlier child behavioral inhibition and attachment, assessed at 14 months, was also considered. Results: Compared to children of non-anxious mothers, children of mothers with social phobia were significantly more likely to give anxiously negative responses in their school DP (OR = 2.57). In turn, negative DP predicted teacher reported anxious-depressed and social worry problems. There were no effects of infant behavioral inhibition or attachment. Conclusion: Vulnerability in young children at risk of anxiety can be identified using Doll Play narratives

Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase

Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

<p>Abstract</p> <p>Background</p> <p>T-cell based immunotherapy for lung cancer (LC) could be a promising and novel therapeutic approach. Six-transmembrane epithelial antigen of the prostate (STEAP) and the polycomb group protein enhancer of zeste homolog 2 (EZH2) are highly expressed in LC and since the expression of molecules in normal tissue is significantly lower as compared to tumor cells, these proteins are considered as potential tumor-associated antigens (TAAs) for developing T-cell based immunotherapy.</p> <p>Methods</p> <p>We assessed the capacity of predicted CD4 T-cell epitopes from STEAP and EZH2 to induce anti-tumor immune responses to LC cell lines.</p> <p>Results</p> <p>Out of several predicted epitopes, two synthetic peptides, STEAP_281-296and EZH2_95-109, were effective in inducing CD4 T-cell responses that were restricted by HLA-DR1, DR15, or DR53 molecules, indicating that the peptides function as promiscuous T-cell epitopes. Moreover, STEAP_281-296and EZH2_95-109-reactive T-cells could directly recognize STEAP or EZH2 expressing LC cells in an HLA-DR restricted manner. In addition, some STEAP-reactive T-cells responded to STEAP+ tumor cell lysates presented by autologous dendric cells. Most significantly, both of these peptides were capable of stimulating <it>in vitro </it>T-cell responses in patients with LC.</p> <p>Conclusions</p> <p>Peptides STEAP_281-296and EZH2_95-109function as strong CD4 T-cell epitopes that can elicit effective anti-tumor T-cell responses against STEAP or EZH2 expressing LC. These observations may facilitate the translation of T-cell based immunotherapy into the clinic for the treatment of LC.</p

EZH2 Codon 641 Mutations are Common in BCL2-Rearranged Germinal Center B Cell Lymphomas

Mutations at codon 641 of EZH2 are recurrent in germinal center B cell lymphomas, and the most common variants lead to altered EZH2 enzymatic activity and enhanced tri-methylation of histone H3 at lysine 27, a repressive chromatin modification. As an initial step toward screening patients for cancer genotype-directed therapy, we developed a screening assay for EZH2 codon 641 mutations amenable for testing formalin-fixed clinical specimens, based on the sensitive SNaPshot single nucleotide extension technology. We detected EZH2 mutations in 12/55 (22%) follicular lymphomas (FL), 5/35 (14%) diffuse large B cell lymphomas with a germinal center immunophenotype (GCB-DLBCL), and 2/11 (18%) high grade B cell lymphomas with concurrent rearrangements of BCL2 and MYC. No EZH2 mutations were detected in cases of Burkitt lymphoma (0/23). EZH2 mutations were frequently associated with the presence of BCL2 rearrangement (BCL2-R) in both the FL (28% of BCL-R cases versus 0% of BCL2-WT cases, p<0.05) and GCB-DLBCL groups (33% of BCL2-R cases versus 4% of BCL2-WT cases, p<0.04), and across all lymphoma types excluding BL (27% of BCL2-R cases versus 3% of BCL2-WT cases, p<0.003). We confirmed gain-of-function activity for all previously reported EZH2 codon 641 mutation variants. Our findings suggest that EZH2 mutations constitute an additional genetic “hit” in many BCL2-rearranged germinal center B cell lymphomas. Our work may be helpful in the selection of lymphoma patients for future trials of pharmacologic agents targeting EZH2 and EZH2-regulated pathways

Mimicry of Food Intake: The Dynamic Interplay between Eating Companions

Numerous studies have shown that people adjust their intake directly to that of their eating companions; they eat more when others eat more, and less when others inhibit intake. A potential explanation for this modeling effect is that both eating companions' food intake becomes synchronized through processes of behavioral mimicry. No study, however, has tested whether behavioral mimicry can partially account for this modeling effect. To capture behavioral mimicry, real-time observations of dyads of young females having an evening meal were conducted. It was assessed whether mimicry depended on the time of the interaction and on the person who took the bite. A total of 70 young female dyads took part in the study, from which the total number of bites (N = 3,888) was used as unit of analyses. For each dyad, the total number of bites and the exact time at which each person took a bite were coded. Behavioral mimicry was operationalized as a bite taken within a fixed 5-second interval after the other person had taken a bite, whereas non-mimicked bites were defined as bites taken outside the 5-second interval. It was found that both women mimicked each other's eating behavior. They were more likely to take a bite of their meal in congruence with their eating companion rather than eating at their own pace. This behavioral mimicry was found to be more prominent at the beginning than at the end of the interaction. This study suggests that behavioral mimicry may partially account for social modeling of food intake