We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcus species (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens), Streptococcus agalactiae (4 antigens), and Propionibacterium acnes (4 antigens) were selected by comparative immunoproteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% for S. agalactiae, and 38.5%/84.8% for P. acnes. The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values.
The dual-specificity tyrosine phosphorylated and regulated kinase Mirk/DYRK1B belongs to an evolutionary conserved family of kinases involved in the control of growth and development. DYRK1B has low level of expression in most normal cell types but is amplified or over-expressed in a number of human cancers. DYRK1B regulates the exit of cancer cells from quiescence through regulating cyclin D turnover and p27kip1 stabilization, thus participating as substantial actor in the control of cancer cell cycle progression. Diaxonhit has developed a novel class of DYRK inhibitors with potent in vitro efficacy. Among them, EHT 5372 reduces tumor growth in a PANC-1 xenograft model and reduces ascites spheroids to single cells and induces their apoptosis. Three-dimensional (3D) multicellular spheroids are symmetrical cellular aggregates that model an in vitro system of intermediate complexity between monolayer cultures and tumors in vivo. Here, we have conducted genome-wide analysis of transcriptional changes between normal (monolayer, 2D) and 3D culture conditions of pancreatic PANC-1 cells using Diaxonhit's GWSATM platform to identify tumor-relevant genes and pathways. The PANC-1 multicellular spheroid model was first characterized based on phenotypical and functional properties and we then analyzed the pharmacological response of spheroids to EHT 5372 and other DYRK inhibitors treatment. A strong up-regulation of DYRK1B was evidenced and confirmed at the protein level. Stemness-related markers were investigated and some were also found much higher in the spheroids than within the monolayer cultures and further increased in a culture time-dependent manner. Stem-like cancer cells may be the cause of therapy-resistance and relapse in patients and sphere forming ability is one of properties of this quiescent cancer-initiating cells. As DYRK1B contributes to G0 arrest to maintain the viability of quiescent cancer cells, pharmacological DYRK inhibition would reduce the capacity of cells to enter into quiescence and sensitize cancer cells to conventional chemotherapeutic agents or radiation. Molecular and physiological consequences of inhibiting DYRK1B with EHT 5372 are now being investigated along with genes expression changes in stemness-related markers and in biological pathways involved in tumor cell growth regulation. A molecular characterization of pancreatic cancer cell spheroids is currently undertaken to determine the utility of the 3D assay as a surrogate tool enriched in cancer stem cells. Spheroids also enable the study of quiescence, chemoresistance and metastasis. DYRK1B inhibitors screening in 3D models is a powerful approach that can help focus on compounds active on these crucial mechanisms of cancer cell biology. The most active compounds identified in this program are likely good candidates for in vivo xenograft studies. Citation Format: Anne-Sophie CASAGRANDE, Florence BACHELOT, Emeline THROO, Florence MAHE, Bertrand LEBLOND, Thierry BESSON, Matthew PANDO, Laurent DESIRE. 3D multicellular pancreatic cancer spheroids as drug screening tool for pharmacological evaluation of EHT 5372 and other Mirk/DYRK1B inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2620. doi:10.1158/1538-7445.AM2014-2620
Abnormal alternative splicing occurs in cancer, resulting in the production of novel transcript variants. Understanding the diverse mechanisms by which splicing dysregulation contributes to human disease will generate new perspectives for drug development and biomarkers identification. The overall aim of this work is the generation of libraries of alternative splicing events that are deregulated in cancer and during anti-cancer treatments. The goal of these libraries is to be interrogated for the identification of novel biomarkers, allowing to monitor disease status, progression/relapse, and specificity/selectivity of drug response. The platform used is Exonhit's Genome Wide SpliceArray, a new generation of microarray that extends transcriptomic profiling to the monitoring of alternative splicing, thereby increasing the discriminatory power of the analyses. Here, we profiled different cancers to identify novel targets and markers that are either commonly regulated across multiple cancers or specific of a given cancer type. Alternatively spliced transcripts were isolated from breast, colon, and lung tumors and their corresponding adjacent normal tissues (20 each). Different splicing patterns were evidenced in tumoral versus normal tissues and from specificity analysis performed across a pool of 20 normal organs. Events of interest, focused on splicing events that generate potential novel amino acid sequences, were selected based on combination of statistical analysis of probe sets deregulations, protein knowledge and pathway analyses. The events were subsequently validated by QPCR analysis in these 3 cancers. These validated events will be used to identify novel cell surface epitopes for antibody development with therapeutic or diagnostic usefulness. Following this same approach, we also performed a comparative analysis of the transcriptomic response of endothelial cells to tubulin polymerization inhibitors, in order to test the feasibility of identifying differential drug response markers. To do so, we profiled various tubulin inhibitors in an in vitro angiogenesis inhibition assay. HUVEC cells were induced to form neovessels in vitro in the presence or absence of the tested tubulin inhibitors. Then, microarray profiling was performed, followed by a detailed analysis with Ingenuity, focusing on up-regulated genes with biomarker potential. Our results demonstrate that alternative RNA splicing offers a currently underexploited source of biological information for cancer research. Platforms dedicated to alternative splicing such as the SpliceArray can be integrated into discovery processes to allow identification of novel targets for drug discovery and biomarkers identification. Further, such platforms may also provide guidance in the selection and follow-up of patients in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B29.
Alternative RNA splicing is a key molecular mechanism for the generation of functional protein diversity. Abnormal alternative splicing can occur in cancer, resulting in the production of novel transcript variants. Therefore, development of platform technologies aimed at studying alternative splicing will help to understand the diverse mechanisms by which splicing dysregulation contribute to human disease. The impact of alternative splicing in disease and its regulation by drug actions have opened up new perspectives for target discovery, drug development and drug response monitoring. ExonHit has generated this type of discovery platform, the Genome Wide SpliceArrayTM, and is currently building libraries of alternative splicing events that are deregulated in cancer and in cases of therapy resistance. These libraries can be interrogated to answer questions such as drug response monitoring, specificity and selectivity of drug response, diagnostics for monitoring disease status, progression or relapse. Using ExonHit's SpliceArrayTM, we previously reported a significant contribution of splicing to cancer development and drug response (1). Here, we demonstrated the capability of ExonHit's platforms 1/ to identify novel cell surface epitopes for antibody development and 2/ to study drug resistance. Based on SpliceArray™ microarray analysis, transcripts alternatively spliced were isolated from breast tumor tissues for the epitope study. Different splicing patterns were evidenced in tumoral versus normal tissues and specificity analysis was performed too. Based on combination of statistical analysis and protein knowledge, most relevant events were selected as alternatively spliced transcripts encoding novel amino acid sequence that can be used to target monoclonal antibodies. Finally, QPCR expression analysis validate the specificity of the selected events identified by the probe sets that emerged from the genome-wide splicing analysis. For the drug resistance study, two types of Imatinib-resistant leukemia cell lines were established. Distinct splicing patterns were evidenced using principal component analysis, through statistical analysis of differential splicing and pathway analyses. We also implemented selectivity filters allowing to identify a drug resistance signature and a set of pathways/genes involved in drug resistance. These signatures will need to be confirmed in subsequent studies. Our results demonstrate that alternative RNA splicing offers a currently underexploited source of novel disease targets. Platforms dedicated to studying alternative splicing can be integrated into discovery processes to allow identification of novel targets for drug discovery and we have began to identify genomic expression profiles related to drug treatment that will ultimately help monitoring drug response and clinical trial design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4860. doi:10.1158/1538-7445.AM2011-4860
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