Gene expression profiling of breast tumours from New Zealand patients

AIMS: New Zealand has one of the highest rates of breast cancer incidence in the world. We investigated the gene expression profiles of breast tumours from New Zealand patients, compared them to gene expression profiles of international breast cancer cohorts and identified any associations between altered gene expression and the clinicopathological features of the tumours. METHODS: Affymetrix microarrays were used to measure the gene expression profiles of 106 breast tumours from New Zealand patients. Gene expression data from six international breast cancer cohorts were collated, and all the gene expression data were analysed using standard bioinformatic and statistical tools. RESULTS: Gene expression profiles associated with tumour ER and ERBB2 status, molecular subtype and selected gene expression signatures within the New Zealand cohort were consistent with those found in international cohorts. Significant differences in clinicopathological features such as tumour grade, tumour size and lymph node status were also observed between the New Zealand and international cohorts. CONCLUSIONS: Gene expression profiles, which are a sensitive indicator of tumour biology, showed no clear di¬fference between breast tumours from New Zealand patients and those from non-New Zealand patients. This suggests that other factors may contribute to the high and increasing breast cancer incidence in New Zealand compared to international populations

Brise Du Sud

80.7568.169 – “Brise Du Sud”: Eug. Chassaignac/ P. Canonge: Bronze Pen Print: 1864: Vocal Solo

The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa

The aim of this study was to investigate the relationship between seminal leukocytes, reactive oxygen species (ROS) production in the ejaculate, and markers of apoptosis in human spermatozoa. Semen samples were collected from 60 patients attending fertility clinics at the Reproductive Biology Unit at Tygerberg Academic Hospital and Vincent Pallotti Hospital, Cape Town, South Africa. The concentration of seminal leukocytes was determined and was correlated with ROS production in the ejaculate, the percentage of superoxide (·O2 )- and hydrogen peroxide (H2O2)-positive spermatozoa, glutathione activation in the ejaculate, and with markers of apoptosis in spermatozoa, namely cysteine-dependent aspartate-directed proteases (caspase)-3/7 activation, mitochondrial membrane potential (ΔΨm), and the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive sperm. Significant correlations with the concentration of seminal leukocytes were found for ROS production in the ejaculate, the percentage of ·O2 -positive spermatozoa, and caspase-3/7 activation in the ejaculate. Leukocytospermic samples showed significantly higher ROS production, percentage of ·O2 -positive sperm, GSH activation, and caspase-3/7 activation compared to non-leukocytospermic samples. The percentage of ·O2 -positive sperm was significantly correlated with sperm ΔΨm and caspase-3/7 activation in the ejaculate. Sperm ΔΨm and TUNEL-positive sperm did not correlate with seminal leukocyte concentration. Data demonstrate that high seminal leukocyte concentrations that leads to increased seminal ROS production, and is also associated with caspase activation in the male germ cell and increased mitochondrial ROS production. The latter could possibly be a result of disturbed ΔΨm. The activation of caspase-3/7 could then follow the increased intrinsic superoxide levels due to depleted intrinsic glutathione (GSH). These cellular events might not directly and immediately lead to DNA fragmentation as an endpoint of apoptosis because of topological hindrances.Web of Scienc

Zinc Finger Nuclease mediated knockout of ADP dependent Glucokinase in Cancer cell lines: Effects on cell survival and Mitochondrial Oxidative Metabolism

<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div

Accessing a New Dimension in TP53 Biology: Multiplex Long Amplicon Digital PCR to Specifically Detect and Quantitate Individual TP53 Transcripts

TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53's alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5' and 3' ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.fals

The Development of Inclusive Learning Relationships in Mainstream Settings: A Multimodal Perspective

The debate regarding the inclusion of children with Special Educational Needs and Disabilities (SEND) in mainstream education in the UK partly revolves around what makes the classroom environment inclusive. Through the potential offered by the specific qualitative methodologies employed, this study aimed to explore the development of teachers’ pedagogical practices and learning relationships upon the inclusive education of children with special educational needs and disabilities in two primary school classes. The study considered the views and behaviours of primary school pupils with and without special educational needs, primary school teachers and teaching assistants (TAs) in one mainstream school. Drawing on a multimodal approach to discourse analysis to account for the complex relationships between symbolic and non-verbal modes of classroom signification, the study explored how meaning is produced in classrooms and children’s modes of communication, as well as in teachers’ practices. The two classes are compared on the basis of teaching observations, interviews, transcripts of dialogues, and analyses of classroom organisation and decoration. This paper suggests that the greatest influence on the educational and social outcomes of students with special educational needs is the behaviour and practices of the classroom teacher

Education for citizenship in South Australian public schools: a pilot study of senior leader and teacher perceptions

Preparing students for informed and active citizenship is a core goal of education and schooling in Australia. The ways schools educate and prepare young Australians for citizenship involves a range of processes and initiatives central to the work of schools, including school ethos, mission, extra-curricular activities and community-based participation. With regard to the formal curriculum, the recent introduction and implementation of the first ever Federal Australian curriculum includes provision for a new subject – Civics and Citizenship. Research evidence from other nations suggests that schools understand, approach and enact education for citizenship in a multitude of ways, yet how Australian schools construct this aspect of their work is currently under-researched. In this context, and drawing on data from interviews with school leaders and teachers of year six-eight (11-14 year olds) students in a small sample of South Australian primary and secondary schools, we explore perceptions and current approaches to education for citizenship. Our findings suggest (i) that while school leaders and teachers value education for citizenship, they do so for different reasons; (ii) that schools place values as central to education for citizenship; and, (iii) that community involvement is typically understood as occurring within rather than beyond the school

Tumor mutational burden is a determinant of immune-mediated survival in breast cancer

Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P < 0.001) but not in those with low TMB (TMB-Lo, P = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P = 0.047; TMB-Lo, P = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P = 0.011; TMB-Lo, P = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.fals