HER family receptor activation and dimerisation in colorectal cancer and cancer-derived exosomes

The EGFR pathway is upregulated in several human cancers including colorectal and head and neck cancers. The anti-EGFR antibodies cetuximab and panitumumab are currently widely used in the management of metastatic colorectal cancer with limited duration of response and almost inevitable development of resistance. HER receptors are able to form alternative dimers and can therefore compensate the loss of function of one receptor during targeted therapies. Usage of pan-HER inhibitor such as AZD8931 has been trialed and may represent one way of overcoming resistance to anti-EGFR therapy. However the effects of anti-HER therapy in patients cannot be easily measured without tissue biopsy. Analysis of exosomes offers a potential platform for the longitudinal monitoring of HER signalling in the form of a liquid biopsy by examining their protein and miRNA contents. Foster Resonance Energy Transfer (FRET) assay using fluorescence lifetime imaging microscopy (FLIM) was used for quantitative analysis of HER dimerisation in colorectal cancer cells. Treatment with cetuximab resulted in increase in HER3 phosphorylation and HER2-HER3 heterodimerisation in the sensitive cell line LIM1215, but not in resistant line DLD1 (KRAS WT). Treatment with AZD8931 resulted in reduced phosphorylation in HER1, HER2 and HER3 in both cell lines, and similar HER dimerisation changes as with treatment with cetuximab. HER3 activation and HER2-HER3 dimer rewiring upon anti-HER therapy could contribute to treatment resistance in colorectal cancer cells. Cell-derived and circulating exosomes were isolated using differential ultracentrifugation, and characterised using Nanosight and immunostaining. Reverse phase protein array provided an alternative high-throughput platform to dot blot for exosomal protein analysis but requires further optimisation. Exosomal HER protein quantity reflected HER expression in vitro. In a cohort of patients with advanced colorectal cancer patients undergoing first line systemic therapy, circulating exosomal HER3 changes predicted treatment failure prior to radiological progression. In another cohort of patients receiving first line AZD8931, early changes in exosomal HER2-HER3 dimer may be predictive of response to anti-HER therapy. Furthermore, levels of exosomal miR-21 could be used for monitoring of treatment response in patients with colorectal cancer

PANTHER: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003

HER2-HER3 heterodimer quantification by FRET-FILM and patient subclass analysis of the COIN colorectal trial

BACKGROUND: The phase 3 MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by 'FLIM Histology' in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy +/-cetuximab. Bayesian latent class analysis (LCA) and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation and cetuximab on progression-free survival (PFS) and overall survival (OS). All statistical tests were two-sided. RESULTS: LCA on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS: 1624 days [95%CI=1466-1816] vs 461 [95%CI=431-504]): Class 1 (15.6%) showed a benefit from cetuximab in OS (HR = 0.43 [95%CI=0.25-0.76]; p = 0.004). Class 2 showed an association of increased HER2-HER3 with better OS (HR = 0.64 [95%CI=0.44-0.94]; p = 0.02). A class prediction signature was formed and tested on an independent validation cohort (N = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (N = 1,630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment

Investigation and optimization on melting performance of a triplex-tube heat storage tank by rotational mechanism

Phase change heat storage is the backbone of energy storage technology, but its storage time is affected by the low thermal conductivity of phase change materials. Therefore, the melting performance of a triplex-tube latent heat thermal energy storage unit (T-LHTESU) in a phase change heat storage system is studied in this paper, and the rotation mechanism is applied to the unit. Firstly, a numerical model of the T-LHTESU considering the rotation mechanism is constructed, and the validity of the rotation unit is verified by comparison with experimental data. In this unit, N-eicosane is used as a phase change material for heat exchange. The effects of different rotational speeds on the liquid phase distribution, temperature distribution, flow velocity distribution, total energy storage, and energy storage efficiency of the T-LHTESU are studied. The results show that the melting time of this unit at 0.1 and 1 rpm is 46.98 and 69.35% lower than that of the stationary model, respectively. The total amount of stored heat is decreased by 0.67 and 2.17%, and the heat storage efficiency is increased by 87.34% and 219.19%, respectively. This indicates that the addition of the rotation mechanism greatly increases the heat storage efficiency of the T-LHTESU and reduces its total melting time, while the reduction of the total energy stored in the melting cycle is small. Then it is proved that rotation improves the single heat transfer mechanism of the stationary model and eliminates the thermal deposition caused by natural convection by studying the internal temperature/velocity response of the stationary model and the speed of 0.1 rpm. The related geometric structure of the model is optimized by response surface optimization design based on 0.1 rpm rotation speed. The influence of each variable on the target response is obtained, and compared with the original model, the melting time of the optimized model is reduced by 12.24%. Finally, based on the geometric optimization design, the influence of element physical factors (temperature and material of fin/tube wall) on the related melting properties is studied. This study is helpful to promote the effective use of rotation mechanism in phase change heat storage systems and has a certain guiding role in the structural design

Isothermal kinase-triggered supramolecular assemblies as drug sensitizers

Protein kinases, the main regulators of a vast map of cellular processes, are the most attractive targets in drug discovery. Despite a few successful examples of protein kinase inhibitors, the drug discovery strategy of downregulating protein kinase activity has been quite limited and often fails even in animal models. Here, we utilize protein kinase A (PKA) activity to design PKA-triggered supramolecular assemblies with anticancer activities. Grafting a suitable peptide to PNIPAM raises the critical temperature of the LCST polymer above body temperature. Interestingly, the corresponding phosphorylated polymer has a critical temperature below body temperature, making this peptide-appended PNIPAM a suitable polymer for the PKA-triggered supramolecular assembly process. PKA-triggered assembly occurs selectively in PKA-upregulated MCF-7 cells, which disturbs the cytoskeleton and sensitizes cancer cells against doxorubicin. The chemosensitization is also observed in vivo to identify effective tumor inhibitors with satisfactory biocompatibility. Overall, this phosphorylation-induced (in principle, PKA-catalyzed) supramolecular assembly opens up a promising chemotherapy strategy for combating kinase-upregulated cancer

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin-magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV

Trace MicroRNA Quantification by Means of Plasmon-Enhanced Hybridization Chain Reaction

Quantifying trace microRNAs (miRNAs) is extremely important in a number of biomedical applications but remains a great challenge. Here we present an enzyme-free amplification strategy called plasmon-enhanced hybridization chain reaction (PE-HCR) for quantifying trace miRNAs with an outstanding linear range from 1 fM to 1 pM (<i>r</i>² = 0.991), along with a detection limit of 0.043 fM (1300 molecules in 50 μL of sample). The merits of the PE-HCR assay, including high sensitivity and specificity, quantitative detection, no enzyme involvement, low false positives, and easy-to-operate procedures, have been demonstrated for high-confidence quantification of the contents of miRNAs in even single cancer cells. The PE-HCR assay may open up new avenues for highly sensitive quantification of biomarkers and thus should hold great potentials in clinical diagnosis and prognosis

Microdroplet digital PCR:Detection and quantitation of biomarkers in archived tissue and serial plasma samples in patients with lung cancer

IntroductionThere is much interest in the use of noninvasive biomarkers in the management of lung cancer, particularly with respect to early diagnosis and monitoring the response to intervention. Cell-free tumor DNA in patients with cancer has been shown to hold potential as a noninvasive biomarker, in which the response to treatment may be evaluated using a blood test only. Multiple technologies have been suggested as being appropriate to measure cell-free tumor DNA. Microdroplet digital polymerase chain reaction (mdPCR) has a number of attributes that suggest it may be a useful tool for detecting clinically relevant genetic events. It offers precise and accurate quantitation of mutant alleles, including rare variants.MethodsWe evaluate the performance of mdPCR in the analysis of DNA extracted from reference standards, tumor biopsies, and patient plasma.ResultsThe potential of mdPCR to detect clinically relevant mutations is demonstrated, in both formalin-fixed paraffin-embedded material and plasma. Furthermore, we show that mdPCR can be used to track changes in peripheral blood biomarkers in response to treatment and to detect the emergence of drug-resistant clones.ConclusionsMdPCR has potential as a tool to detect and quantify tumor-derived mutational events in cell-free DNA from patients with lung cancer