Quantitation of endogenous metabolites in mouse tumors using mass-spectrometry imaging

Described is a quantitative-mass-spectrometryimaging (qMSI) methodology for the analysis of lactate and glutamate distributions in order to delineate heterogeneity among mouse tumor models used to support drug-discovery efficacy testing. We evaluate and report on preanalysisstabilization methods aimed at improving the reproducibility and efficiency of quantitative assessments of endogenous molecules in tissues. Stability experiments demonstrate that optimum stabilization protocols consist of frozen-tissue embedding, post-tissue-sectioning desiccation, and storage at −80 °C of tissue sections sealed in vacuum-tight containers. Optimized stabilization protocols are used in combination with qMSI methodology for the absolute quantitation of lactate and glutamate in tumors, incorporating the use of two different stable-isotope-labeled versions of each analyte and spectral-clustering performed on each tissue section using k-means clustering to allow region-specific, pixel-by-pixel quantitation. Region-specific qMSI was used to screen different tumor models and identify a phenotype that has low lactate heterogeneity, which will enable accurate measurements of lactate modulation in future drug-discovery studies. We conclude that using optimized qMSI protocols, it is possible to quantify endogenous metabolites within tumors, and region-specific quantitation can provide valuable insight into tissue heterogeneity and the tumor microenvironment

Family functioning, parental psychological distress and child behaviours : evidence from the Victorian child health and wellbeing study

We examined, using data from the 2006 Victorian Child Health and Wellbeing Study (VCHWS), whether family functioning is associated with parental psychological distress and children&rsquo;s behavioural difficulties. The VCHWS was a statewide cross-sectional telephone survey to 5,000 randomly selected primary caregivers of 0- to 12-year-old children between October 2005 and March 2006. Only parents or guardians of children aged 4&ndash;12 years (n = 3,370) were included in this study. After adjusting for sociodemographic variables and ethnicity, parents or guardians scoring higher on the family functioning scale (i.e., from poorly functioning households) were at greater risk of psychological distress and had children with lower levels of prosocial behaviour and higher levels of behavioural difficulties relative to those from healthily functioning households. Mental health prevention programmes addressing child mental and conduct problems should consider the family environment and target those families functioning poorly.<br /

Investigating anhedonia in a non-conventional species: Do some riding horses Equus caballus display symptoms of depression?

International audienceInvestigating depression-like conditions in animals is methodologically challenging, but potentially important for welfare. Some riding horses display ‘withdrawn’ states of inactivity and low responsiveness that resemble the reduced engagement with the environment shown by certain depressed patients. To assess whether these animals are experiencing a depression-like state, we investigated anhedonia – the loss of pleasure, a key symptom of human depression – in 20 withdrawn and non-withdrawn horses from the same stable. The time horses spent being withdrawn appeared unrelated to age or sex, but correlated with time devoted to stereotypic behaviour, a possible marker of lifetime stress. Comparison with data collected 5 years earlier also revealed that horses scored as withdrawn then remained significantly likely to display the behaviour. We measured sucrose intake, a classic measure of anhedonia never previously applied to horses. Flavoured sugar blocks, novel to these subjects, were mounted in each stall and weighed 3, 8, 24 and 30 h after provision. We predicted that if affected by depression-like states, the most withdrawn horses would consume the least sucrose. This prediction was met (F1,18 = 4.65, two tailed p = 0.04). This pattern could, however, potentially reflect general appetite levels and/or food neophobia. To control for these confounds, hay consumption was measured over 5 days, as were subjects’ latencies to eat a meal scented with a novel odour. Although low hay consumption and long latencies to eat scented food did predict low sucrose consumption, statistically controlling for these confounds did not eliminate the relationship between being withdrawn and consuming less sucrose (although reducing it to a strong trend): F1,15 = 4.28, two-tailed p = 0.056. These data thus suggest long-lasting depression-like states in certain riding horses, which correlate with stereotypic behaviour and are characterised by anhedonia and bouts of ‘withdrawn’ unresponsiveness

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle. SIGNIFICANCE: We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented. (C) 2014 AACR.

Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of Both Sensitizing and T790M Resistance Mutations That Spares the Wild Type Form of the Receptor

Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile