Inhibition of EGFR, HER2, and HER3 signalling in patients with colorectal cancer wild-type for BRAF, PIK3CA, KRAS, and NRAS (FOCUS4-D): a phase 2–3 randomised trial

Background: A substantial change in trial methodology for solid tumours has taken place, in response to increased understanding of cancer biology. FOCUS4 is a phase 2–3 trial programme testing targeted agents in patients with advanced colorectal cancer in molecularly stratified cohorts. Here, we aimed to test the hypothesis that combined inhibition of EGFR, HER2, and HER3 signalling with the tyrosine kinase inhibitor AZD8931 will control growth of all wild-type tumours. Methods: In FOCUS4-D, we included patients from 18 hospitals in the UK with newly diagnosed advanced or metastatic colorectal cancer whose tumour was wild-type for BRAF, PIK3CA, KRAS, and NRAS. After 16 weeks of first-line therapy, patients with stable or responding tumours were randomised to oral AZD8931 (40 mg twice a day) or placebo. Randomisation was done by minimisation with a random element of 20%, minimisation by hospital site, site of primary tumour, WHO performance status, 16-week CT scan result, number of metastatic sites, and first-line chemotherapy regimen. The primary outcome was progression-free-survival. CT scans were assessed by local radiologists according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Preplanned interim analyses were assessed per protocol and were agreed using multiarm multistage (MAMS) trial design methodology triggered by occurrence of progression-free survival events in the placebo group. The final analysis was assessed by intention to treat. This trial is registered at , ISRCTN 90061546. Findings: Between July 7, 2014, and March 7, 2016, 32 patients were randomised to study treatment, 16 to AZD8931 and 16 to placebo. At the first preplanned interim analysis (March, 2016), the independent data monitoring committee (IDMC) recommended closure of FOCUS4-D because of a lack of activity. At the final analysis (Aug 1, 2016), 31 patients had had a progression-free survival event (15 with AZD8931 and 16 with placebo). Median progression-free survival was 3·48 months (95% CI 1·51–5·09) in the placebo group and 2·96 months (1·94–5·62) in the AZD8931 group. No progression-free survival benefit of AZD8931 compared with placebo was noted (hazard ratio [HR] 1·10, 95% CI 0·47–3·57; p=0·95). The most common grade 3 adverse event in the AZD8931 group was skin rash (three [20%] of 15 patients with available data vs none of 16 patients in the placebo group), and in the placebo group it was diarrhoea (one [7%] vs one [6%]). No grade 4 adverse events were recorded and no treatment-related deaths were reported. Interpretation: The MAMS trial design for FOCUS4 has shown efficiency and effectiveness in trial outcome delivery, informing the decision to proceed or stop clinical evaluation of a targeted treatment within a molecularly defined cohort of patients. The overarching FOCUS4 trial is now aiming to open a replacement arm in the cohort with all wild-type tumours. Funding: Medical Research Council (MRC) and National Institute for Health Research (NIHR) Efficacy and Mechanism Evaluation programme, Cancer Research UK, NIHR Clinical Trials Research Network, Health and Care Research Wales, and AstraZeneca

Use of a genetically engineered mouse model as a preclinical tool for HER2 breast cancer

Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies presents a major clinical problem. Although preclinical studies have identified a number of possible mechanisms, clinical validation has been difficult. This is most likely to reflect the reliance on cell-line models that do not recapitulate the complexity and heterogeneity seen in human tumours. Here, we show the utility of a genetically engineered mouse model of HER2-driven breast cancer (MMTV-NIC) to define mechanisms of resistance to the pan-HER family inhibitor AZD8931. Genetic manipulation of MMTV-NIC mice demonstrated that loss of phosphatase and tensin homologue (PTEN) conferred de novo resistance to AZD8931, and a tumour fragment transplantation model was established to assess mechanisms of acquired resistance. Using this approach, 50% of tumours developed resistance to AZD8931. Analysis of the resistant tumours showed two distinct patterns of resistance: tumours in which reduced membranous HER2 expression was associated with an epithelial-to-mesenchymal transition (EMT) and resistant tumours that retained HER2 expression and an epithelial morphology. The plasticity of the EMT phenotype was demonstrated upon re-implantation of resistant tumours that then showed a mixed epithelial and mesenchymal phenotype. Further AZD8931 treatment resulted in the generation of secondary resistant tumours that again had either undergone EMT or retained their original epithelial morphology. The data provide a strong rationale for basing therapeutic decisions on the biology of the individual resistant tumour, which can be very different from that of the primary tumour and will be specific to individual patients

Picture book exposure elicits positive visual preferences in toddlers

Although the relationship between "mere exposure" and attitude enhancement is well established in the adult domain, there has been little similar work with children. This article examines whether toddlers' visual attention toward pictures of foods can be enhanced by repeated visual exposure to pictures of foods in a parent-administered picture book. We describe three studies that explored the number and nature of exposures required to elicit positive visual preferences for stimuli and the extent to which induced preferences generalize to other similar items. Results show that positive preferences for stimuli are easily and reliably induced in children and, importantly, that this effect of exposure is not restricted to the exposed stimulus per se but also applies to new representations of the exposed item. (C) 2009 Elsevier Inc. All rights reserved

Src family kinase inhibitor saracatinib (AZD0530) impairs oxaliplatin uptake in colorectal cancer cells and blocks organic cation transporters

Elevated Src Family Kinase (SFK) activity is associated with tumour invasion and metastasis. The SFK inhibitor saracatinib (AZD0530) is currently in Phase II trials in patients including those with colorectal cancer (CRC), where links between SFK activity and poor prognosis are particularly striking. Saracatinib is likely to be used clinically in combination regimens, specifically with 5FU and oxaliplatin in CRC. The aim of this study was to determine the impact of saracatinib on oxaliplatin and 5FU efficacy in CRC cells. Saracatinib did not modulate 5FU efficacy but antagonized oxaliplatin in a schedule specific manner through reduced oxaliplatin uptake via an SFK independent mechanism. Saracatinib resembles the pharmacophore of known organic cation transporter (OCT) inhibitors and reduced oxaliplatin efficacy maximally in cells over-expressing OCT2. These data suggest that oxaliplatin uptake in CRC is attenuated by saracatinib via inhibition of OCT2, a potential consideration for the clinical development of this SFK inhibitor

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein

AbstractThe eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2,3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals

Phosphorus and sulfur metabonomic profiling of tissue and plasma obtained from tumour-bearing mice using ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry

RATIONALE: Metabonomic studies use complex biological samples (blood plasma/serum, tissues, etc.) that when analysed with high-performance liquid chromatography/mass spectrometry (HPLC/MS) or nuclear magnetic resonance (NMR) generate profiles that may contain many thousands of features. These profiles can be difficult to interpret with the majority of the features contributing little to the study. As such there is an argument for the development of techniques that can simplify the problem by targeting particular classes of compounds. METHODS: In this study ultra-performance liquid chromatography/inductively coupled plasma mass spectrometry (UPLC/ICP-MS) was used to profile tumour tissue and plasma samples for phosphorus- and sulfur-containing metabolites. These samples were xenograft tumours (derived from breast, lung and colon cell lines) and plasma obtained from nude mice. Plasma was also obtained from non-tumour-bearing mice as a control. Due to isobaric interferences this method took advantage of the dynamic reaction cell within the ICP-MS system to react the phosphorus and sulfur ions with oxygen. The PO+ and SO+ ions were then monitored free of interferences. The total phosphorus and sulfur within each sample was also recorded using flow injection ICP-MS. A robust quality control system based on pooled sample replicate analysis was used throughout the study. RESULTS: Determination of the total phosphorus and sulfur content of each sample was sufficient in itself for statistical differentiation between the majority of the cell lines analysed. Subsequent reversed-phase chromatographic profiling of the organic tumour and plasma extracts revealed the presence of a number of well-retained phosphorus-containing compounds that showed tumour-specific profiles. Reversed-phase profiling was not suitable for the sulfur-containing compounds which eluted with the solvent front. CONCLUSIONS: This study has shown the potential use of UPLC/ICP-MS to differentiate between tumour cell lines, using both plasma and tumour tissue samples, based solely on metabolites that contain phosphorus or sulfur. Whilst further work is required to identify these compounds this methodology shows the ability of the described methods to provide targets for future biomarker discovery studies