Comparative analysis of drug response and gene profiling of HER2-targeted tyrosine kinase inhibitors.

BACKGROUND: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations. METHODS: In this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data. RESULTS: All three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity in HER2-mutant and EGFR-mutant cells. High expression of HER2, VTCN1, CDK12, and RAC1 correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance. BRCA2 mutations were correlated with neratinib and tucatinib response, and high expression of ATM, BRCA2, and BRCA1 were associated with neratinib resistance. CONCLUSIONS: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

Combined Cellular and Biochemical Profiling to Identify Predictive Drug Response Biomarkers for Kinase Inhibitors Approved for Clinical Use between 2013 and 2017.

Kinase inhibitors form the largest class of precision medicine. From 2013 to 2017, 17 have been approved, with 8 different mechanisms. We present a comprehensive profiling study of all 17 inhibitors on a biochemical assay panel of 280 kinases and proliferation assays of 108 cancer cell lines. Drug responses of the cell lines were related to the presence of frequently recurring point mutations, insertions, deletions, and amplifications in 15 well-known oncogenes and tumor-suppressor genes. In addition, drug responses were correlated with basal gene expression levels with a focus on 383 clinically actionable genes. Cell lines harboring actionable mutations defined in the FDA labels, such as mutant BRAF(V600E) for cobimetinib, or ALK gene translocation for ALK inhibitors, are generally 10 times more sensitive compared with wild-type cell lines. This sensitivity window is more narrow for markers that failed to meet endpoints in clinical trials, for instance CDKN2A loss for CDK4/6 inhibitors (2.7-fold) and KRAS mutation for cobimetinib (2.3-fold). Our data underscore the rationale of a number of recently opened clinical trials, such as ibrutinib in ERBB2- or ERBB4-expressing cancers. We propose and validate new response biomarkers, such as mutation in FBXW7 or SMAD4 for EGFR and HER2 inhibitors, ETV4 and ETV5 expression for MEK inhibitors, and JAK3 expression for ALK inhibitors. Potentially, these new markers could be combined to improve response rates. This comprehensive overview of biochemical and cellular selectivities of approved kinase inhibitor drugs provides a rich resource for drug repurposing, basket trial design, and basic cancer research.

Cell Panel Profiling Reveals Conserved Therapeutic Clusters and Differentiates the Mechanism of Action of Different PI3K/mTOR, Aurora Kinase and EZH2 Inhibitors.

Cancer cell line panels are important tools to characterize the in vitro activity of new investigational drugs. Here, we present the inhibition profiles of 122 anticancer agents in proliferation assays with 44 or 66 genetically characterized cancer cell lines from diverse tumor tissues (Oncolines). The library includes 29 cytotoxics, 68 kinase inhibitors, and 11 epigenetic modulators. For 38 compounds this is the first comparative profiling in a cell line panel. By strictly maintaining optimized assay protocols, biological variation was kept to a minimum. Replicate profiles of 16 agents over three years show a high average Pearson correlation of 0.8 using IC50 values and 0.9 using GI50 values. Good correlations were observed with other panels. Curve fitting appears a large source of variation. Hierarchical clustering revealed 44 basic clusters, of which 26 contain compounds with common mechanisms of action, of which 9 were not reported before, including TTK, BET and two clusters of EZH2 inhibitors. To investigate unexpected clusterings, sets of BTK, Aurora and PI3K inhibitors were profiled in biochemical enzyme activity assays and surface plasmon resonance binding assays. The BTK inhibitor ibrutinib clusters with EGFR inhibitors, because it cross-reacts with EGFR. Aurora kinase inhibitors separate into two clusters, related to Aurora A or pan-Aurora selectivity. Similarly, 12 inhibitors in the PI3K/AKT/mTOR pathway separated into different clusters, reflecting biochemical selectivity (pan-PI3K, PI3Kßγδ-isoform selective or mTOR-selective). Of these, only allosteric mTOR inhibitors preferentially targeted PTEN-mutated cell lines. This shows that cell line profiling is an excellent tool for the unbiased classification of antiproliferative compounds. Mol Cancer Ther; 15(12); 3097-109. ©2016 AACR.

Application of elements of microbiological risk assessment in the food industry via a tiered approach.

Food safety control is a matter for concern for all parts of the food supply chain, including governments that develop food safety policy, food industries that must control potential hazards, and consumers who need to keep to the intended use of the food. In the future, food safety policy may be set using the framework of risk analysis, part of which is the development of (inter)national microbiological risk assessment (MRA) studies. MRA studies increase our understanding of the impact of risk management interventions and of the relationships among subsequent parts of food supply chains with regard to the safety of the food when it reaches the consumer. Application of aspects of MRA in the development of new food concepts has potential benefits for the food industry. A tiered approach to applying MRA can best realize these benefits. The tiered MRA approach involves calculation of microbial fate for a product and process design on the basis of experimental data (e.g., monitoring data on prevalence) and predictive microbiological models. Calculations on new product formulations and novel processing technologies provide improved understanding of microbial fate beyond currently known boundaries, which enables identification of new opportunities in process design. The outcome of the tiered approach focuses on developing benchmarks of potential consumer exposure to hazards associated with new products by comparison with exposure associated with products that are already on the market and have a safe history of use. The tiered prototype is a tool to be used by experienced microbiologists as a basis for advice to product developers and can help to make safety assurance for new food concepts transparent to food inspection services.

Estimating the probability of recontamination via the air using Monte Carlo simulations.

Recontamination of food products can cause foodborne illnesses or spoilage of foods. It is therefore useful to quantify this recontamination so that it can be incorporated in microbiological risk assessments (MRA). This paper describes a first attempt to quantify one of the recontamination routes: via the air. Data on the number of airborne microorganisms were collected from literature and industries. The settling velocities of different microorganisms were calculated for different products by combining the data on aerial concentrations with sedimentation counts assuming that settling is under the influence of gravity only. Air movement is not explicitly considered in this study. Statistical analyses were performed to clarify the effect of different products and seasons on the number of airborne microorganisms and the settling velocity. For both bacteria and moulds, three significantly different product categories with regard to the level of airborne organisms were identified. The statistical distribution in these categories was described by a lognormal distribution. The settling velocity did not depend on the product, the season of sampling or the type of microorganism, and had a geometrical mean value of 2.7 mm/s. The statistical distribution of the settling velocity was described by a lognormal distribution as well. The probability of recontamination via the air was estimated by the product of the number of bacteria in the air, the settling velocity, and the exposed area and time of the product. For three example products, the contamination level as a result of airborne recontamination was estimated using Monte Carlo simulations. What-if scenarios were used to exemplify determination of design criteria to control a specified contamination level.

Physiological and mathematical aspects in setting criteria for decontamination of foods by physical means.

In heat processing, microbial inactivation is traditionally described as log-linear. As a general rule, the relation between rate of inactivation and temperature is also described as a log-linear relation. The model is also sometimes applied in pressure and in pulsed electric field (PEF) processing. The model has proven its value by the excellent safety record of the last 80 years, but there are many deviations from log-linearity. This could lead to either over-processing or under-processing resulting in safety problems or, more likely, spoilage problems. As there is a need for minimal processing, accurate information of the inactivation kinetics is badly needed. To predict inactivation more precisely, models have been developed that can cope with deviations of linearity. As extremely low probabilities of survival must be predicted, extrapolation is almost always necessary. However, extrapolation is hardly possible without knowledge of the nature of nonlinearity. Therefore, knowledge of the physiology of inactivation is necessary. This paper discusses the physiology of denaturation by heat, high pressure and pulse electric field. After discussion of the physiological aspects, the various aspects of the development of inactivation models will be addressed. Both general and more specific aspects are discussed such as choice of test strains, effect of the culture conditions, conditions during processing and recovery conditions and mathematical modelling of inactivation. In addition to lethal inactivation, attention will be paid to sublethal inactivation because of its relevance to food preservation. Finally, the principles of quantitative microbiological risk assessment are briefly mentioned to show how appropriate inactivation criteria can be set.