Assay Readout Research Articles

Evidence for Highly Variable, Region-Specific Patterns of T-Cell Epitope Mutations Accumulating in Mycobacterium tuberculosis Strains.

Vaccines that confer protection through induction of adaptive T-cell immunity rely on understanding T-cell epitope (TCE) evolution induced by immune escape. This is poorly understood in tuberculosis (TB), an ancient, chronic disease, where CD4 T-cell immunity is of recognized importance. We probed 905 functionally validated, curated human CD4 T cell epitopes in 79 Mycobacterium tuberculosis (Mtb) whole genomes from India. This screen resulted in identifying 64 mutated epitopes in these strains initially using a computational pipeline and subsequently verified by single nucleotide polymorphism (SNP) analysis. SNP based phylogeny revealed the 79 Mtb strains to cluster to East African Indian (EAI), Central Asian Strain (CAS), and Beijing (BEI) lineages. Eighty-nine percent of the mutated T-cell epitopes (mTCEs) identified in the 79 Mtb strains from India has not previously been reported. These mTCEs were encoded by genes with high nucleotide diversity scores including seven mTCEs encoded by six antigens in the top 10% of rapidly divergent Mtb genes encoded by these strains. Using a T cell functional assay readout, we demonstrate 62% of mTCEs tested to significantly alter CD4 T-cell IFNγ and/or IL2 secretion with associated changes in predicted HLA-DR binding affinity: the gain of function mutations displayed higher predicted HLA-DR binding affinity and conversely mutations resulting in loss of function displayed lower predicted HLA-DR binding affinity. Most mutated antigens belonged to the cell wall/cell processes, and, intermediary metabolism and respiration families though all known Mtb proteins encoded mutations. Analysis of the mTCEs in an SNP database of 5,310 global Mtb strains identified 82% mTCEs to be significantly more prevalent in Mtb strains isolated from India, including 36 mTCEs identified exclusively in strains from India. These epitopes had a significantly higher predicted binding affinity to HLA-DR alleles that were highly prevalent in India compared to HLA-DR alleles rare in India, highlighting HLA-DR maybe an important driver of these mutations. This first evidence of region-specific TCE mutations potentially employed by Mtb to escape host immunity has important implications for TB vaccine design.

Abstract IA27: Oncolytic viruses: Potential for in situ anti-tumor vaccination and combination with checkpoint blockade

Abstract Oncolytic virotherapy (OV) is increasingly recognised as a form of immunotherapy, with the lead agent, a herpes simplex virus (HSV) encoding GM-CSF, talimogene laherparepvec (T-Vec), now licensed for clinical use by intratumoural injection in advanced melanoma. Although early clinical data is showing promise for OV/immune checkpoint blockade combinations, the underlying mechanisms by which such strategies may be optimised for the widest possible clinical application, remain poorly understood. We have focused on intravenous delivery of OV as a more practical clinical approach than intratumoural injection, and shown that the dsRNA, non-genetically modified OV reovirus, can access tumours in patients in the brain or liver following systemic injection, despite the presence of anti-viral neutralising antibodies (NAB) in the blood prior to treatment. The ability of reovirus (but not necessarily all OV) to evade antibody-mediated neutralisation appears to be via protective cell carriage (or "hitchhiking") of virus or virus/NAB complexes by monocytes in the circulation. Once systemically delivered reovirus has accessed the brain tumour microenvironment we showed, consistent with clinical data using intratumoural T-Vec in melanoma, that the virus converted an immunologically ‘cold’ tumour milieu to ‘hot’, which effectively primed for combination oncolytic immunovirotherapy with immune checkpoint blockade, when tested back in an immunocompetent murine glioma model. To complement murine data and translational clinical trial sample analysis, we have developed human in vitro pre-clinical models of innate and adaptive anti-tumour immune priming by OV. OV innately activated human peripheral blood mononuclear cells (PBMC), including differentiation of monocytes towards a more antigen presenting cell (APC)-like phenotype. Hence OV-hitchhiking monocytes in the blood, after delivering virus to tumour, may pick up infected tumour cells undergoing immunogenic cell death and act as APC to initiate adaptive anti-tumour immune priming. In support of this paradigm, we showed that human monocytes, antigen loaded by phagocytosis of OV-infected dying tumour cells, supported greater priming of a specific anti-tumour CD8+ cytotoxic T cell (CTL) response than uninfected controls, when co-cultured with responder PBMC. This OV-mediated enhanced anti-tumour CTL priming response included expansion of T cells specific for multiple, non-mutated tumour associated antigens (TAA) expressed by the original, antigen loading tumour cell, as demonstrated using a CTL re-stimulation readout assay. In this assay, autologous monocytes were first pulsed with long, 15mer overlapping peptides, spanning the length of 3 melanoma TAAs, gp100, TRP-2 or MART-1. MHC class I epitopes processed from these long peptides pools, and presented by the monocytes, could then stimulate their specific, responder T cells on co-culture with primed CTL, and the TAA-specific, non-HLA restricted T cell response quantified by FACS staining for intracellular IFN gamma and CD8 or CD4. Using this assay, we showed that both reovirus and HSV-infected tumour cells primed a stronger anti-TAA CTL response than their uninfected controls. This priming was further enhanced if the antigen load tumour cells were treated with a histone deacetylase inhibitor, which induced expression of otherwise silent TAA, as well as HSV.Taken together, these data support the use of OV, including via systemic administration, as a platform to trigger immunogenic tumour cell death and in-situ vaccination, across both human and mouse systems, to both enhance the response to current immunotherapy using checkpoint blockade, and to suggest novel combinations incorporating other immunomodulatory drugs. Citation Format: Alan Melcher. Oncolytic viruses: Potential for in situ anti-tumor vaccination and combination with checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA27.

A smartphone readout system for gold nanoparticle-based lateral flow assays: application to monitoring of digoxigenin

For modern approaches in precision medicine, fast and easy-to-use point-of-care diagnostics (POCs) are essential. Digoxin was chosen as an example of a drug requiring close monitoring. Digoxin is a cardiac glycoside used for the treatment of tachycardia with a narrow therapeutic window of 0.5–2.0 ng·mL−1, and toxic effects are common for concentrations above 2.5 ng·mL−1. For monitoring of blood concentration levels and treatment of intoxication, highly selective antibodies for digoxin and its hapten, digoxigenin, are available. A smartphone readout system is described for measuring digoxigenin in human serum using a common gold nanoparticle lateral flow assay (LFA). The R-package GNSplex, which also includes a Shiny app for quantitative test interpretation based on linear models, is used for image analysis. Images of lateral flow strips were taken with an iPhone camera and a simple darkbox made from black cardboard. Sensitivity and accuracy of the quantitative smartphone system as well as analytical parameters such as limit of detection (LOD) were determined and compared to data obtained with a high resolution BioImager. The data show that the smartphone based digoxin assay yields reliable quantitative results within the clinically relevant concentration range.Graphical abstractFor therapeutic drug monitoring and point of care diagnostics we introduce the open source R-package GNSplex for smartphone readout and interpretation of lateral flow assays. The cardiac glycoside dixogin was used as target for this quantitative smartphone reader

Analysis of antibody responses to selected Plasmodium falciparum merozoite surface antigens in mild and cerebral malaria and associations with clinical outcomes.

Merozoite surface proteins (MSPs) are critical for parasite invasion; they represent attractive targets for antibody-based protection against clinical malaria. To identify protection-associated target MSPs, the present study analysed antibody responses to whole merozoite extract (ME) and to defined MSP recombinant antigens in hospitalized patients from a low endemic urban area as a function of disease severity (mild versus cerebral malaria). Sera from 110 patients with confirmed severe cerebral malaria (CM) and 91 patients with mild malaria (MM) were analysed (mean age=29years) for total and subclass immunoglobulin (Ig)G to ME and total IgG to MSP1p19, MSP2, MSP3, MSP4 and MSP5 by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were evaluated using the antibody-dependent respiratory burst (ADRB) assay in a subset of sera. There was a trend towards higher IgG1 and IgG4 levels to ME in CM compared to MM; only ME IgM responses differed significantly between fatal and surviving CM patients. Increased prevalence of IgG to individual MSPs was found in the CM compared to the MM group, including significantly higher levels of IgG to MSP4 and MSP5 in the former. Sera from fatal (24·5%) versus surviving cases showed significantly lower IgG to MSP1p19 and MSP3 (P<0·05). ADRB assay readouts correlated with high levels of anti-MSP IgG, and trended higher in sera from patients with surviving compared to fatal CM outcome (P=0·07). These results document strong differential antibody responses to MSP antigens as targets of protective immunity against CM and in particular MSP1p19 and MSP3 as prognostic indicators.

Identification of Functional Protein Regions Through Chimeric Protein Construction.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides

The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as potential neurotoxic effects. The in vivo metabolites of some chemicals including organophosphorus pesticides can become more potent AChE inhibitors compared to their parental compounds. To account for the effects of biotransformation, we have developed and characterized a high-throughput screening method for identifying AChE inhibitors that become active or more potent following xenobiotic metabolism. In this study, an enzyme-based assay was developed in 1536-well plates using recombinant human AChE combined with human or rat liver microsomes. The AChE activity was measured by two methods with different readouts: colorimetric and fluorescent. The assay exhibited exceptional performance characteristics including large assay signal window, low well-to-well variability and high reproducibility. The performance of the assays with microsomes was characterized by testing a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition. Both assay readouts were required for maximal sensitivity. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity.

Establishment and Analysis of a 3D Co-Culture Spheroid Model of Pancreatic Adenocarcinoma for Application in Drug Discovery.

The high attrition rate of oncology drug candidates can be in part explained by the disconnect between the standard preclinical models (e.g., 2D culture, xenograft tumors) commonly employed for drug discovery and the complex multicellular microenvironment of human cancers. As such, significant focus has recently shifted to the establishment of preclinical models that more closely recapitulate human tumors, such as patient-derived xenografts, 3D spheroids, humanized mice, and mixed-culture models. For these models to be suited to drug discovery, they should optimally exhibit reproducibility, high-throughput, and robust and simple assay readouts. In this article, we describe a protocol for the generation of an in vitro 3D co-culture spheroid model that recapitulates the interaction of tumor cells with stromal fibroblasts in pancreatic adenocarcinoma. We additionally describe protocols relevant to the analysis of these spheroids in high-throughput drug discovery campaigns such as the assessment of spheroid proliferation, immunofluorescence and immunohistochemistry staining of spheroids, live-cell and confocal imaging and analysis of cell surface markers.

Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection.

The development of a high-performance assay readout using integrated detectors is a current challenge in the implementation of DNA tests in diagnostic laboratories, particularly for supporting pharmacogenetic tests. A method for allelic discrimination, associated with single nucleotide polymorphisms (SNPs), is presented. Genomic DNA is extracted from blood and buccal swab samples. The procedure comprises fast multiplex ligation-dependent probe amplification, PCR amplification using universal primers and subsequent barcode hybridization. In this last step, each product is recognized by the specific probes immobilized on the surface of an optical disc. Assay results can be obtained with a disc reader. The optical sensing method in a DNA microarray format was optimized and evaluated for the simultaneous identification of 28 polymorphisms associated with psychiatric pharmacogenomics. The target biomarkers were located in the genes related to drug-metabolizing enzymes and drug transporters. The multiplexing capability and assay selectivity strongly depended on correct design (ligation probes, tails and barcodes). The discriminant analysis of reader outputs (spot intensities) led to patients being classified into different allelic populations. The obtained assignations correlated properly with the results provided by the reference technique (bead arrays), and the assay ended in an 8-fold shorter time using affordable equipment. The combination of a highly selective genotyping reaction as array-MLPA and the compact disc technology provides a reliable point-of-care approach. This genotyping tool is useful for the selection of personalized drug therapies in decentralized clinical laboratories.

Optimized plasma-assisted bi-layer photoresist fabrication protocol for high resolution microfabrication of thin-film metal electrodes on porous polymer membranes

Structured metal thin-film electrodes are heavily used in electrochemical assays to detect a range of analytes including toxins, biomarkers, biological contaminants and cell cultures using amperometric, voltammetric and impedance-based (bio)sensing strategies as well as separation techniques such as dielectrophoresis. Over the last decade, thin-film electrodes have been fabricated onto various durable and flexible substrates including glass, silicon and polymers. However, the combination of thin-film technology with porous polymeric substrates frequently used for biochips often results in limited resolution and poor adhesion of the metal thin-film, thus severely restricting reproducible fabrication and reliable application in e.g. organ-on-a-chip systems. To overcome common problems associated with micro-structured electrode manufacturing on porous substrates, we have optimized a bi-layer lift-off method for the fabrication of thin-film electrodes on commercial porous polyester membranes using a combination of LOR3A with AZ5214E photoresists. To demonstrate practical application of our porous electrode membranes for trans-epithelial electrical resistance measurements a tetrapolar biosensing set-up was used to eliminate the artificial resistance of the porous polymer membrane from the electrochemical recordings. Furthermore, barrier resistance of Bewo trophoblast epithelial cells was compared to a standard Transwell assay readout using a EVOM2 volt-ohm meter.•Bi-layer photo resist lift-off yields resolution down to 2.5 μm.•Argon Plasma-assisted lift-off results in improved adhesion of gold thin films and eliminates the need for chromium adhesion layers.•Membrane electrodes can be used for elimination of the porous membrane resistance during tetra-polar epithelial resistance measurements.

TCR validation toward gene therapy for cancer.

The speed of T cell receptor (TCR) discovery has been revolutionized by barcode-based TCR sequencing approaches that allow the reconstitution of a T cell's paired alpha and beta TCR chain, and the process of TCR discovery promises to become ever faster and cheaper with the continuing development single cell analysis techniques. This technological progress has generated an urgent need to develop efficient TCR validation platforms for the rapid and safe clinical translation of TCRs into therapeutic agents. Whereas much attention has in the past focused on CD8-positive cytotoxic T cells recognizing MHC class I presented epitopes, the increasing demand to validate TCRs expressed on neoepitope-reactive CD4 T cells requires the implementation of large-scale T cell activation-based readout assays to complement existing multimer and cytotoxicity-based assays. Here, we present commonly used TCR validation assays, and include detailed guidance on TCR synthesis, delivery, and appropriate experimental control TCRs. We also comment on upcoming methods that hold promise for further speeding the process of TCR validation, hastening the translation of TCRs from the laboratory into the clinic.

Isotopic resonance at 370\u2009ppm deuterium negatively affects kinetics of luciferin oxidation by luciferase

Since 1930s, it has been known that some biochemical and biological processes exhibit abnormal kinetics at a deuterium concentration in the local environment of 250–600 ppm, which is 2–4 times higher that the normal concentration of 150 ppm D. We sought to test if the kinetics of firefly luciferase oxidizing luciferin, the reaction widely used as a read-out in various biochemical assays, is also affected by an elevated deuterium content. To this end, both luciferase and luciferin substrate solutions were prepared based on water with extra deuterium added to a concentration ranging from 150 ppm and up to 10,000 ppm (1%). Upon mixing the solutions, the luminescence intensity at different times was compared with that of the corresponding control solutions with 150 ppm D. A broad negative resonance was detected (p < 10−6), with a ≈20% drop in luminescence at 370 ppm D. Given that, on average, about half of hydrogen atoms in proteins are not exchangeable in solution, this value corresponds to ≈260 ppm of deuterium in all enzyme’s hydrogens, in a very good agreement with the prediction of the Isotopic resonance hypothesis.

Reproducing the human mucosal environment ex vivo

The medical management of inflammatory bowel disease (IBD) remains problematic with a pressing need for innovation in drug development as well as delivery of personalized therapies. Both the disease's inherent pathophysiologic complexity and heterogeneity in its etiology conspire in making it difficult to accurately model for either the purposes of basic research or drug development. Multiple attempts at creating meaningful experimental models have fallen short of adequately recapitulating the disease and most do not capture any aspect of the cause or the effects of patient heterogeneity that underlays most of the difficulties faced by physicians and their patients. In vivo animal models, tissue culture systems, and more recent synthetic biology approaches are all too simplistically reductionist for the task. However, ex vivo culture platforms utilizing patient biopsies offer a system that more closely mimics end-stage disease processes that can be studied in detail and subjected to experimental manipulations. Recent studies describe further optimization of mucosal explant cultures in order to increase tissue viability and maintain a polarized epithelial layer. Current applications of the platform include studies of the interplay between the epithelial, immune and stromal compartment of the intestinal tissue, investigation of host-microbial interactions, preclinical evaluation of candidate drugs and uncovering mechanisms of action of established or emerging treatments for IBD. Patient explant-based assays offer an advanced biological system in IBD that recapitulates disease complexity and reflects the heterogeneity of the patient population. In its current stage of development, the system can be utilized for drug testing prior to the costlier and time-consuming evaluation by clinical trials. Further refinement of the technology and establishment of assay readouts that correlate with therapeutic outcomes will yield a powerful tool for personalized medicine approaches in which individual patient responses to available treatments are assessed a priori, thus reducing the need for trial and error within the clinical setting.

Technical and diagnostic performance of a new fully automated immunoassay for the determination of intact fibroblast growth factor 23 (FGF23)

There is growing interest in measuring plasma fibroblast growth factor 23 (FGF23) concentrations in a number of clinical settings. However, a reliable assay with acceptable performance is lacking. Plasma samples of healthy adults and patients with different stages of chronic kidney disease (CKD) were used to compare the precision, recovery, linearity and the pre-analytical stability characteristics of a new fully automated FGF23 (intact) assay with a commercially available FGF23 (intact) ELISA. Method agreement was evaluated, reference and stage-specific ranges for kidney disease were established. Other biomarkers relevant for CKD were measured and compared with the FGF23 assays. The fully automated FGF23 (intact) assay demonstrated superior performance compared with the ELISA. A marked positive proportional bias was detected relative to the ELISA assay readout, especially in samples of higher concentration of patients undergoing hemodialysis. Overall, the method comparison revealed a poor degree of correlation. A significant inverse correlation was found between the glomerular filtration rate and both FGF23 assays (both p < .001). Regression analysis revealed that both assays are suitable to predict progression of CKD. A positive correlation was found between FGF23 and phosphate, parathyroid hormone (PTH) and vitamin D, 25(OH)D and 1,25(OH)2D-total assays, respectively. Cutoff points between different stages of CKD were calculated by receiver operator characteristic analysis. The fully automated assay displayed an improved discrimination compared with the ELISA, especially in mild to moderate kidney disease. The new fully automated FGF23 (intact) assay demonstrates excellent analytical performance data and represents a robust, fast and precise alternative to manual FGF23 testing.

Bleomycin Revisited: A Direct Comparison of the Intratracheal Micro-Spraying and the Oropharyngeal Aspiration Routes of Bleomycin Administration in Mice.

Idiopathic Pulmonary Fibrosis (IPF) is a fatal disease characterized by exuberant deposition of extracellular matrix components, deterioration of lung architecture and impairment of lung functions. Its etiopathogenesis remains incompletely understood, as reflected in the lack of an appropriate therapy. Modeling the human disease in mice via the administration of bleomycin (BLM), despite the inherent limitations, has provided valuable insights into the underlying pathogenetic mechanisms, and has been instrumental for the development and validation of new pharmacologic interventions. Here we have directly compared the, most widely used, intratracheal (IT) route of administration with oropharyngeal aspiration (OA). Our results suggest that the OA route of BLM-administration can be used as a safe and effective alternative, minimizing peri-operative and experimental mortality, while preserving a solid fibrotic profile, as assessed with a plethora of standardized readout assays.

Anti-virulence potential of eugenol-rich fraction of Syzygium aromaticum against multidrug resistant uropathogens isolated from catheterized patients

Considering the emergence of biofilm-associated pathogens with multidrug resistance, the objective(s) of the present study was to evaluate the anti-virulence property of Syzygium aromaticum extracts/fractions against 2 multidrug-resistant catheter isolates. Pulverized clove buds were subjected to bioactivity-guided isolation to identify the bioactive extract/fraction(s) with significant anti-virulence property. The clove bud powder was subjected to Soxhlet extraction and all these extracts were investigated in terms of anti-virulent efficacy using initial readout assays. Bioassay-guided partial purification was performed through column and size exclusion chromatography. Detailed testing of the anti-virulence and anti-biofilm activity of sub-minimum inhibitory concentration (sub-MIC) levels of the active fraction, was performed besides the cytotoxicity profiling in RAW 264.7 cells. Gas chromatography-mass spectrometry (GC-MS) analysis of the clove bioactive fraction-2 (CBF-2), done after the bioassay-guided fractionation, revealed eugenol as the major bioactive ingredient present in CBF-2. Reduced mRNA levels of virulence factor genes after CBF-2 (700 μg/ml) treatment correlated well with the respective phenotypic assays (p<0.001). Similarly, CBF-2 (700 μg/ml) treatment exhibited significantly low mRNA levels of quorum sensing (QS) receptor genes compared to their cognate synthase genes (p<0.001). Crystal violet staining and scanning electron micrographs of CBF-2-treated biofilms showed lesser macrocolonies with remarkably simplified architecture. Cytotoxic evaluation of CBF-2 suggested a minute reduction in viability even at the highest tested concentration (1600 μg/ml, p<0.05). The present study indicated that eugenol-rich CBF-2 has potent anti-virulence and anti-biofilm activity against urinary catheter isolates and can be regarded as an alternative for treatment of catheter-associated urinary tract infections.

Abstract 1616: Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting

Abstract Assays measuring protein complexes represent a novel class of protein-based biomarkers. Using proximity-based readouts, we and others have previously shown utility in monitoring signaling-associated complexes to predict response to targeted therapeutics in lung cancer. However, fluorescence-based proximity assay readouts are difficult to implement in a clinical setting and are not conducive to typical pathology laboratory workflow. Here, we provide evidence of a novel proximity-based technology platform that utilizes a “caged hapten” proximity readout followed by chromogenic immunohistochemistry (IHC) detection. We have synthesized haptens modified with a phosphorylated quinone methide precursor. This enzyme- labile caging group abrogates antibody binding; however, upon exposure to alkaline phosphatase (AP) the native hapten is regenerated. These caged haptens may be applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the protein complex population, can then be visualized by an anti-hapten antibody conjugated to horseradish peroxidase (HRP) followed by 3,3'-diaminobenzidine (DAB) detection. These reagents and assays are fully automated on the VENTANA DISCOVERY ULTRA platform. We demonstrate detection of β-Catenin:E-Cadherin, EGFR:GRB2 and MET:GRB2 complexes in formalin-fixed paraffin embedded (FFPE) lung cancer cell lines, patient-derived xenografts (PDX) and non-small cell lung cancer (NSCLC) patient specimens. Assay specificity was confirmed through technical controls involving reagent omission experiments, and biologically by treatment with small molecule kinase inhibitors that disrupt kinase:adaptor interactions. We revealed heterogeneous EGFR and MET signaling in MET-amplified PDX specimens, replicating previously-published findings. For EGFR:GRB2, we developed a pathologist-guided ordinal scoring system and evaluated inter-rater concordance using a tissue microarray containing 120 NSCLC cores. Kappa statistics and inter-rater correlations reveal excellent correlation among multiple raters.Collectively, these data support the adaptation of proximity-based approaches in clinical pathology settings. The use of caged hapten proximity assays on VENTANA automated slide stainers provides a novel and robust platform to evaluate protein complexes as potential biomarkers that may be incorporated as molecular correlates in ongoing early phase clinical trials. This approach also facilitates analysis of a large number of slides in a reproducible assay format enabling retrospective evaluation of tumor specimens from completed trials. Citation Format: Brian D. Kelly, Nathan W. Polaske, Yuri Belosludtsev, Matthew A. Smith, Theresa Boyle, Y. Ann Chen, Eric B. Haura. Novel caged hapten proximity assay platform enables detection of oncogenic signaling-associated complexes in the clinical pathology laboratory setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1616.

Abstract 5867: Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, representing 8% of all childhood tumors. The most aggressive subtype of RMS is associated with chromosomal translocations creating a fusion transcription factor, most commonly involving PAX3 and FOXO1 (fusion-positive (FP)-RMS). Our group has recently comprehensively characterized the genetic and epigenetic landscape of FP-RMS. We found that FP-RMS has a strikingly low somatic mutational burden and that the PAX3-FOXO1 fusion gene is the primary oncogenic driver. Specifically, we found that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super enhancers in collaboration with the bromodomain and extra-terminal domain (BET) protein family member BRD4, and the master transcription factors MYOG, MYOD1, and MYCN. We therefore hypothesized that FP-RMS would be vulnerable to disruption of the PAX3-FOXO1 super enhancer oncogenic circuitry. Method and Results: To test this, we developed a novel readout assay to simultaneously monitor PAX3-FOXO1 super enhancer and general transcriptional activity as well as cell viability, and screened against a 62,643 small-molecule library at a concentration of 10 µM. We found a total of 573 compounds with selective ALK super enhancer inhibition, which were selected for dose response assay with four 10-fold dilutions ranging from 20 μM to 2 nM. Further prioritization using a systematic computational model reduced this number to 64. Among the compounds with known activities, the multi-kinase PKC-412/midostaurin was previously identified in screens for PAX3-FOXO1 inhibitors. Other known compounds included topoisomerase I and topoisomerase II inhibitors, where both enzymes have recently been reported to play important roles in BRD4-facilitated DNA dynamics involving super enhancers and the transcriptional machinery. Among this list we identified 33 novel compounds of unknown mechanism of action. We therefore performed RNA-seq analysis to determine the transcriptional impact of the effect of these compounds. Pathway, gene set enrichment and Connectivity Map analyses of RNA-seq expression signatures implicate several of the novel compounds as inhibitors of epigenetic regulators, including inhibitors of BRD4, histone deacetylases, histone demethylases, histone methyltransferases, and chromodomain proteins. Conclusions: Our systematic approach identified several small molecules that specifically disrupt the activity of the PAX3-FOXO1 fusion oncogene in RMS. Further characterization of the specificity and selectivity of the lead compounds is ongoing, and exact mechanism of action is currently being elucidated. Our ultimate goal is to develop a clinically effective precision therapeutic targeting the previously “undruggable” PAX3-FOXO1 oncogenic driver of aggressive FP-RMS. Citation Format: Robert G. Hawley, Girma M. Woldemichael, Berkley E. Gryder, Young Song, Sivasish Sindiri, Vineela Gangalapudi, Jun S. Wei, James B. McMahon, Jack F. Shern, Javed Khan. Identification of novel inhibitors of the PAX3-FOXO1 oncogenic driver in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5867.

Quantitative multiplex one-step RT-PCR assay for identification and quantitation of Sabin strains of poliovirus in clinical and environmental specimens

An improved quantitative multiplex one-step RT-PCR (qmosRT-PCR) for simultaneous identification and quantitation of all three serotypes of poliovirus is described. It is based on using serotype-specific primers and fluorescent TaqMan oligonucleotide probes. The assay can be used for high-throughput screening of samples for the presence of poliovirus, poliovirus surveillance and for evaluation of virus shedding by vaccine recipients in clinical trials to assess mucosal immunity. It could replace conventional methods based on cell culture virus isolation followed by serotyping. The assay takes only few hours, and was found to be simple, specific, sensitive and has large quantitative linearity range. In addition, the method could be used as readout in PCR-based poliovirus titration and neutralization assays.

Exploration of the Fluorescent Properties and the Modulated Activities against Sirtuin Fluorogenic Assays of Chromenone-Derived Natural Products

Chromenone-derived natural products include chromones (flavone, isoflavone) and coumarins. Chromenone compounds not only exhibit impressive biological activities, but also are an important resource of experimentally used fluorophores, such as, 7-amino-4-methylcoumarin (AMC). Various chromenone compounds have reported to have weak fluorescence, and this has the potential to interfere with the measurements during AMC fluorogenic assays and result in non-robust assay readouts. Several flavones and isoflavones were found as SIRT1 activators, while fluorogenic sirtuin assays utilized AMC labelled peptides as the substrates. In this study we investigated whether the fluorescent properties of chromenone-derived natural products interrupt the measurement of SIRT1/2 modulated activities. We found that the reported SIRT1 activators: flavones were detected with the SIRT1 activation activity, but isoflavones were not detected with SIRT1 activation activity, and instead that they were found to be fluorogenic compounds. Another chromenone compound, osthole, exhibited a moderate SIRT2 inhibitory activity with an IC50 of 10 μM. In conclusion, the fluorescent properties of these chromenone compounds do affect the measurement of the sirtuin activities of both inhibitors and activators. However, if the possible fluorescence properties are mitigated in the assay readout, these fluorogenic assays enable the screening of activity modulators.

Uneven Staining in Automated Immunohistochemistry: Cold and Hot Zones and Implications for Immunohistochemical Analysis of Biopsy Specimens.

The occurrence of uneven staining (UES) in automated immunohistochemistry (IHC) has been experienced by clinical laboratories and has the potential to confound readout, interpretation, and reporting of IHC assays despite the presence optimally stained on-slide controls. However, there are no studies of this phenomenon in regard to the type, frequency, and association with different automated IHC platforms. We studied the occurrence of UES in automated IHC assays with real world examples from clinical practice and by using a laboratory developed methodology to monitor baseline and periodic performance of automated IHC instruments. Sections of formalin-fixed, paraffin-embedded normal liver tissue were mounted on 180 glass slides and stained for HepPar1 on 6 automated IHC instruments (4 different models from 3 different manufacturers). Macroscopic and microscopic defects of staining were recorded. Only 8% of slides showed completely uniform staining. UES, including areas of both increased and decreased staining, occurred with all instruments. Decreased staining was often zonal, involving large regions of the slide. Decreased staining mostly localized in an instrument-dependent manner. Increased staining tended to occur in small foci with a random distribution. The common occurrence of UES (particularly decreased staining) has important implications for the reliable read-out of IHC assays on biopsy samples. Baseline and periodic quality assurance testing for UES is recommended for all automated IHC instruments.