Articles de revues : « Predictive functional assay »

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Runser, Anne, Caroline Schaning, Frédéric Allemand et Jean Amiral. « An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia ». Biomedicines 9, n^o 3 (13 mars 2021) : 296. http://dx.doi.org/10.3390/biomedicines9030296.

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Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test.

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Duca, Francesca, Luisa Ruggeri, Guido Finazzi, Barbara Negri, Marco Moia et Monica Galli. « Congenital Resistance to Activated Protein C in Patients with Lupus Anticoagulants : Evaluation of Two Functional Assays ». Thrombosis and Haemostasis 80, n^o 08 (1998) : 246–49. http://dx.doi.org/10.1055/s-0037-1615182.

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SummaryThe R506Q mutation (“Factor V Leiden”) is responsible for the resistance to activated Protein C (aPCR), that is evaluated by coagulation tests. Such tests cannot be used in patients with lupus anticoagulants (LAs), due to the interfering effect exerted by these antibodies on “in vitro” phospholipid-dependent coagulation tests. For this reason, assays have been developed to evaluate aPCR that are insensitive to the presence of LA antibodies. We evaluated two such coagulation tests in the plasma of 82 consecutive patients with LAs. By polymerase chain reaction 3 patients (3.6%) were found heterozygous for the R506Q mutation. aPCR was evaluated by two clotting assays, proposed to be “insensitive” to the presence of LAs: 1. aPCR-tissue factor-based assay, using Factor V deficient plasma and 1:40 diluted test plasma; 2. aPCR-dRVVT-based assay with highly concentrated phospholipids. Their interassay coefficient of variation was 28% and 6.2%, respectively. Compared to the polymerase chain reaction analysis, the 2 tests displayed the following characteristics: sensitivity 67% vs 100%, specificity 92% vs 96%, positive predictive value 25% vs 50%, negative predictive value 99% vs 100%, respectively. Among LA patients without the R506Q mutation, 5 scored positive in the aPCR-tissue factor-based assay, 2 in the aPCR-dRVVT-based assay and another one in both assays. Our findings suggest that the aPCRdRVVT-based test is more reliable and sensitive than the aPCR-tissue factor-based one to the R506Q mutation in patients with LAs. Both assays, when negative, make unlikely the presence of the R506Q mutation. Polymerase chain reaction analysis remains, however, to be performed when either test is positive.

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Hoffman, Gabriel E., Jaroslav Bendl, Kiran Girdhar, Eric E. Schadt et Panos Roussos. « Functional interpretation of genetic variants using deep learning predicts impact on chromatin accessibility and histone modification ». Nucleic Acids Research 47, n^o 20 (23 septembre 2019) : 10597–611. http://dx.doi.org/10.1093/nar/gkz808.

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Abstract Identifying functional variants underlying disease risk and adoption of personalized medicine are currently limited by the challenge of interpreting the functional consequences of genetic variants. Predicting the functional effects of disease-associated protein-coding variants is increasingly routine. Yet, the vast majority of risk variants are non-coding, and predicting the functional consequence and prioritizing variants for functional validation remains a major challenge. Here, we develop a deep learning model to accurately predict locus-specific signals from four epigenetic assays using only DNA sequence as input. Given the predicted epigenetic signal from DNA sequence for the reference and alternative alleles at a given locus, we generate a score of the predicted epigenetic consequences for 438 million variants observed in previous sequencing projects. These impact scores are assay-specific, are predictive of allele-specific transcription factor binding and are enriched for variants associated with gene expression and disease risk. Nucleotide-level functional consequence scores for non-coding variants can refine the mechanism of known functional variants, identify novel risk variants and prioritize downstream experiments.

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Palmer, Jessica A., Alan M. Smith, Vitalina Gryshkova, Elizabeth L. R. Donley, Jean-Pierre Valentin et Robert E. Burrier. « A Targeted Metabolomics-Based Assay Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Identifies Structural and Functional Cardiotoxicity Potential ». Toxicological Sciences 174, n^o 2 (10 février 2020) : 218–40. http://dx.doi.org/10.1093/toxsci/kfaa015.

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Abstract Implementing screening assays that identify functional and structural cardiotoxicity earlier in the drug development pipeline has the potential to improve safety and decrease the cost and time required to bring new drugs to market. In this study, a metabolic biomarker-based assay was developed that predicts the cardiotoxicity potential of a drug based on changes in the metabolism and viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was conducted in 2 phases: (1) biomarker identification and (2) targeted assay development. In the first phase, metabolomic data from hiPSC-CM spent media following exposure to 66 drugs were used to identify biomarkers that identified both functional and structural cardiotoxicants. Four metabolites that represent different metabolic pathways (arachidonic acid, lactic acid, 2′-deoxycytidine, and thymidine) were identified as indicators of cardiotoxicity. In phase 2, a targeted, exposure-based biomarker assay was developed that measured these metabolites and hiPSC-CM viability across an 8-point concentration curve. Metabolite-specific predictive thresholds for identifying the cardiotoxicity potential of a drug were established and optimized for balanced accuracy or sensitivity. When predictive thresholds were optimized for balanced accuracy, the assay predicted the cardiotoxicity potential of 81 drugs with 86% balanced accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing the thresholds for sensitivity yields a balanced accuracy of 85%, 90% sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a paradigm that can identify structural and functional cardiotoxic drugs that could be used in conjunction with other endpoints to provide a more comprehensive evaluation of a drug’s cardiotoxicity potential.

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Park, Sang Hyuk, Chan-Jeoung Park, Dae-Young Kim, Bo-Ra Lee, Young Jin Kim, Young-Uk Cho, Seongsoo Jang et Hyun-Sook Chi. « Evaluation Of Multidrug Resistance (MDR) Functional Activity and Expression Levels Of P-Glycoprotein and MDR Related Protein-1 For The Prediction Of Treatment Failure In Chronic Myeloid Leukemia Patients ». Blood 122, n^o 21 (15 novembre 2013) : 2600. http://dx.doi.org/10.1182/blood.v122.21.2600.2600.

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Abstract Background One of the resistance mechanisms of chronic myeloid leukemia (CML) is multidrug resistance (MDR) mediated by overexpression of P-glycoprotein (P-gp) and MDR related protein-1 (MRP1). The flow cytometric functional assay using efflux inhibitor cyclosporine and Rhodamine-123 (Rho-123 efflux assay) detects MDR functional activity. In this study, we performed Rho-123 efflux assay as well as MRP1 and P-gp expression assays by flow cytometry, and evaluated the performance of each test for the prediction of treatment failure in CML patients. Methods A total of 229 peripheral blood samples (19 diagnosis and 210 follow-up) from 94 CML patients treated with TKIs were enrolled. These samples were categorized into three subgroups [optimal response (N=120), suboptimal response (N=54), treatment failure (N=36)] defined by the response criteria to treatment with TKIs in CML patients (Best Pract Res Clin Haematol 2009;22:331-41), and ABL1 mutation analysis was performed in some patients (N=18) with treatment failure. In addition, cutoff values (mean + 1.65SD) for each test were determined from 35 normal controls. The Rho-123 efflux assay, MRP1 and P-gp expression assays were performed separately with all samples. In the Rho-123 efflux assay, the MDR functional activity was measured as the ratio of mean fluorescence intensity (mean channel fluorescence, mcf) with and without cyclosporine. In the P-gp and MRP1 expression assays, MRP1 and P-gp expression levels were expressed as the positivity (%) with each representative monoclonal antibody. The results were compared between patients with treatment response (optimal and suboptimal) and failure. The sensitivity/specificity/negative predictive values (NPV)/positive predictive values (PPV) of 3 test results were calculated using estimated cutoff values and compared. Results The cutoff values of Rhodamine-123 efflux assay, MRP1, and P-gp expression assays were calculated to be > 1.68, > 4.18%, and > 5.07%, respectively. The patients with treatment failure showed significantly higher MRP1 expression [mean 14.08% vs. 4.48%, P = 0.038 (diagnosis); 5.24% vs. 3.54%, P = 0.006 (follow-up)] and P-gp expression [12.27% vs. 3.41%, P = 0.027 (diagnosis); 5.25% vs. 3.48%, P = 0.005(follow-up)] than those with treatment response. However, there were no significant differences in the Rhodamine-123 efflux assay (P = 0.769 and 0.199 at diagnosis and follow-up) between 2 groups. Subsequent analysis revealed that 58.7% of follow-up patients with treatment failure show positive results in at least one test, and 53.3% of these patients with negative for the ABL1 kinase domain mutation also show positive results in at least one test. The sensitivity/specificity/NPV/PPV of 3 test results for the prediction of treatment failure were estimated as 22.9%/85.1%/84.6%/23.5% (Rhodamine-123 efflux assay), 37.1%/69.7%/83.9%/20.6% (MRP1 expression assay), and 31.4%/78.8%/84.4%/23.9% (P-gp expression assay). Conclusion MRP1 and P-gp expression were significantly higher in follow-up CML patients with treatment failure than those with treatment response. The patients who experienced treatment failure, had shown significantly higher MRP1 and P-gp expression at diagnosis than those who did not. Low sensitivity and PPV are inevitable for assays measuring MDR, because MDR is only one of mechanism that causes treatment failure. Our study revealed relatively high specificity and NPV in both MRP-1 and P-gp expression assay, and at least half of ABL1 mutation negative patients with treatment failure possessed positivity in MDR tests. Therefore, it can be speculated that the estimation of MRP-1 and P-gp expression levels can provide useful informations for the prediction of treatment failure in CML patients. Disclosures: No relevant conflicts of interest to declare.

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Hoppensteadt, Debra A., Josephine Cunanan, Jeanine M. Walenga, Jawed Fareed, Marianne Wilmer et Michael Janssen. « Clinical Evaluation of a New Functional, Plasma-Based Assay for the Detection of Factor V Leiden - the Pefakit® APC-R Factor V Leiden Assay. » Blood 104, n^o 11 (16 novembre 2004) : 3997. http://dx.doi.org/10.1182/blood.v104.11.3997.3997.

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Abstract Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep vein thrombosis (DVT). Over 80% of the APC resistance phenotypes can be explained by the Factor V Leiden (FVL) mutation. This defect is caused by a point mutation in the factor V gene resulting in a replacement of the amino acid Arg 506 by a Gln residue. For patients who are at risk of thrombosis or who have active thrombosis, it is of interest to be able to screen for the FVL mutation in a easy and reliable manner. There are two types of assays for the detection of FVL. The genotype is reliably detected by PCR technology, but this technology is not readily available to all laboratories nor is it inexpensive. The phenotypic expressions of the defect can be identified by plasma-based functional assays, of which several are commercially available. The functional Pefakit® APC-R FVL assay (Pentapharm; Basel, Switzerland) (APC-R) is a new plasma-based clotting assay developed to overcome the limitations of the current assays, including sensitivity and specificity. Additionally, the APC-R assay is more informative as results specify heterozygous / homozygous opposed to a report of normal / abnormal by all other assays. The APC-R assay is designed to specifically act at the level of the prothrombinase complex using a factor V dependent prothrombin activator isolated from the snake venom of the Notechis scutatus. We investigated the phenotypic and genotypic expressions of FVL in patients with cancer and acute symptomatic DVT and/or PE (n=67). Normal healthy volunteers (n=50) collected in-house served as controls. DNA isolated from buffy coats obtained from citrated anticoagulated blood was used to profile FVL using standard PCR probes. Citrated plasma was used to determine APC-R by the functional Pefakit® assay. Of the 67 patients profiled by molecular analysis, 5 (7%) were heterozygous and 1 (1%) was homozygous for the FVL defect. In the normal healthy volunteers, 3 (6%) were heterozygous and 1 (2%) was homozygous for FVL. Using the APC-R functional plasma-based assay, 4 cancer patients (6%) were heterozygous and 2 (3%) were homozygous. In the normal volunteers, 4 (8%) were heterozygous and 1 (2%). was homozygous with the APC-R assay. The APC-R functional plasma-based assay did not miss any FVL patient shown positive by PCR. The heterozygous cancer patients detected with the functional assay were detected as heterozygous with the PRC method; one patient detected as homozygous with the functional method was determined to be heterozygous by PCR. In the normal healthy volunteers, 1 volunteer that was heterozygous by the plasma based assay was negative by PCR. This study demonstrates that the prevalence of the FVL defect in cancer patients with thrombosis is similar to the normal healthy population. Although additional studies to validate the sensitivity/specificity of the APC-R assay and to establish its positive / negative predictive values are needed, the results of this first investigation suggest that the APC-R assay may provide more useful results than other commercially available assays for the detection of FVL. The results of this study showed a good correlation of the functional FVL. APC-R assay with the reference standard molecular PCR method. These data suggest a practical role of this new assay in the clinical laboratory diagnosis of FVL.

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Ross, D. A., S. Lee, V. Reiser, J. Xue, K. Alves, S. Vaidya, A. Kreamer et al. « Multiplexed Assays by High-Content Imaging for Assessment of GPCR Activity ». Journal of Biomolecular Screening 13, n^o 6 (juillet 2008) : 449–55. http://dx.doi.org/10.1177/1087057108317685.

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G-protein-coupled receptors (GPCR) participate in many disease pathways and represent the largest family of therapeutic targets. Thus, great investments are made to discover drugs modulating GPCR-mediated events. Among functional assays for screening GPCRs, the Transfluor® imaging assay is based on redistribution of cytosolic β-arrestin to an activated GPCR and has become widely used in high-content screening. However, assessing Transfluor® alone has limitations: relying on a single mechanistic step of β-arrestin redistribution during GPCR activation, providing no information on the stimulated GPCR's intracellular fate, and using only a single fluorescent color (green fluorescent protein). Taking full advantage of high-content imaging to screen approximately 2000 compounds, the authors multifplexed the Transfluor® assay with an immunofluorescence-based quantification of GPCR internalization. This approach identified and classified 377 compounds interfering with agonist-induced activation of the Transfluor ® assay, receptor internalization, or both. In addition, a subset of compounds was analyzed for their performance across imaging, cell-based calcium release (fluorometric imaging plate reader [FLIPR]), and biochemical receptor binding assays (scintillation proximity assay). This indicated that the imaging assays have even better predictive power for direct inhibition of receptor binding than the FLIPR assay. In conclusion, compounds inducing unique responses can suggest novel mechanisms of action and be used as tools to study GPCR activation and internalization. ( Journal of Biomolecular Screening 2008:449-455)

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Nagler, Michael, et Tamam Bakchoul. « Clinical and laboratory tests for the diagnosis of heparin-induced thrombocytopenia ». Thrombosis and Haemostasis 116, n^o 11 (septembre 2016) : 823–34. http://dx.doi.org/10.1160/th16-03-0240.

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SummaryA rapid diagnostic work-up is required in patients with suspected heparin-induced thrombocytopenia (HIT). However, diagnosis of HIT is challenging due to a number of practical issues and methodological limitations. Many laboratory tests and a few clinical scoring systems are available but the individual characteristics and the diagnostic accuracy of these are hard to appraise. The 4Ts score is a well evaluated clinical assessment tool with the potential to rule out HIT in many patients. Still, it requires experience and is subject to a relevant inter-observer variability. Immunoassays such as enzyme-linked immunosorbent assays or recently developed rapid assays are able to exclude HIT in a number of patients. But, accuracy of immunoassays differs depending on type of assay, threshold, antibody specificity and even manufacturer. Due to a comparatively low positive predictive value, HIT cannot be confirmed by immunoassays alone. In addition, only some of them are immediately accessible, particularly in small laboratories. While functional assays such as the serotonin release assay (SRA) and the heparin-induced platelet activation assay (HIPA) are considered as gold standard for diagnosis of HIT, they require a highly specialised laboratory. In addition, some of them are not adequately evaluated. In clinical practice, we recommend an integrated diagnostic approach combining not only clinical assessment (the 4Ts score) but immunoassays and functional assays as well. We propose a clear diagnostic algorithm supporting clinical decision-making. Furthermore, we provide an overview of all current laboratory techniques for HIT and discuss diagnostic pathways and strategies to reduce diagnostic errors, and future perspectives.

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Neal, Frances, Joanne Arnold, Christine J. Rossant, Sadhana Podichetty, David Lowne, Claire Dobson, Trevor Wilkinson, Caroline Colley, Rob Howes et Tristan J. Vaughan. « Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays ». Journal of Biomolecular Screening 21, n^o 1 (8 octobre 2015) : 24–34. http://dx.doi.org/10.1177/1087057115610070.

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Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.

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Nguyen, Ha T., Tiffany G. Sheu, Vasiliy P. Mishin, Alexander I. Klimov et Larisa V. Gubareva. « Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays ». Antimicrobial Agents and Chemotherapy 54, n^o 9 (28 juin 2010) : 3671–77. http://dx.doi.org/10.1128/aac.00581-10.

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ABSTRACT The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC50s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC50s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC50s between the wild type and the variants, whereas the IC50s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.

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Vinnikov, V. A., et T. V. Rubleva. « Predictors of radiation-induced complications in radiation oncology based on cell survival tests after ex vivo exposure : literature review ». Український радіологічний та онкологічний журнал 29, n^o 1 (29 mars 2021) : 89–118. http://dx.doi.org/10.46879/ukroj.1.2021.89-118.

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Background. Among cancer patients receiving radiotherapy about 5–15 % may have adverse reactions in normal tissues and organs that limit their treatment in a full, originally scheduled regimen. The development of biomarkers and assays for radiation oncology allowing the prediction of patients’ normal tissue toxicity requires a lot of resourses, threfore its current status amd potential directions for future research have to be periodically analyzed and re-evaluated. Purpose – this review summarizes the methodological approaches and developments in the area of functional laboratory assays based on ex vivo cell survival for the prediction of the individual clinical radiosensitivity. Materials and methods. Data for the analysis and systematization were obtained from the full-text articles published in peer review international scientific journals (in English) in 1990–2020, which were selected by the extensive search in PubMed information database and cross references on the topic “Functional cellular tests for intrinsic radiosensitivity to predict adverse radiation effects and radiotherapy complications”. Results. In theory, it might be expected that clonogenic cell survival after ex vivo irradiation can surve as the best individual predictor of radiation toxicity, as it is an integral indicator of cell damage and decline of their regenerative potential. Tendentially, fibroblasts, as a test system for such studies, did not show significant advantages over lymphocytes either in detecting inter-individual variations in the intrinsic cellular radiosensitivity or in predicting clinical radiation toxicity, even for that in skin. It was found that clonogenic cell survival assay, being very time consuming and technically demanding, also suffers from the lack of sensitivity and specificity, essential uncertainty and low reproducibility of the results, and thus is not suitable for the sceening for the abnormal intrinsic radiosensitivity. However, this type of assays is applicable for the radiobiological expertise post factum in individual cases with unexpected, extreme radiation lesions. Radiation-induced lymphocyte apoptosis assay seems to be more promising however still requires further fundamental research for better understanding of its background and more validation studies in order to assess the optimum patient groups, radiotherapy regimens and adverse effects for its confident use in clinical practice. Changes in the regulation of cell cycle check-points (radiationinduced delay) ex vivo can have either positive or inverted association, or no correlation with clinical radiation responses in tissues, thus so far cannot be included in the toolbox of applied radiobiological tests. Conclusions. To date, in the practice of clinical radiobiology, there are no fully validated and standardized functional tests based on the cell survival after ex vivo irradiation, which would allow a sufficiently accurate prediction of adverse radiation effects in normal tissues of radiotherapy patients. In general, ex vivo tests based on the evaluation of only one form of cell death in one cell type are not fully reliable as a “stand alone” assay, because different pathways of cell death probably play different roles and show different dose response within the overal reaction of the irradiated tissue or critical organ. Such tests should become a part of the multiparametric predictive platforms.

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Henniker, Anthony, David Facey, Mark Hertzberg et Emmanuel Favaloro. « Discrimination of von Willebrands Disease (VWD) Subtypes : Direct Comparison of von Willebrand Factor:Collagen Binding Assay (VWF:CBA) with Monoclonal Antibody (MAB) Based VWF-capture Systems ». Thrombosis and Haemostasis 84, n^o 10 (2000) : 541–47. http://dx.doi.org/10.1055/s-0037-1614064.

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SummaryDiscrimination of von Willebrand’s Disease (VWD) subtypes is important since it influences management. Qualitative [ie Type 2A, 2B, 2M] defects exhibit von Willebrand factor (VWF) discordance and give high VWF:Ag to VWF:’activity’ ratios. Classically, VWF:’activity’ is assessed using the VWF:RCof assay. The VWF:CBA is an ELISAbased VWF-functional adhesive assay which has consistently proved to be superior to VWF:RCof. A commercially available monoclonal antibody (MAB) based ELISA assay system claimed to mimic a VWF:RCof-like activity has also been recently described (‘SE’), as has the production and characterisation of a large number [n = 10] of locally generated anti-VWF MAB. In the current study, we have adapted these MAB to in-house ELISA assays to assess their utility for VWD diagnosis and subtype discrimination, and to compare them with other assay systems. Thus, the VWF:CBA, VWF:RCof by agglutination, the SE assay, and in-house MAB based assays have been directly compared for their ability to discriminate Type 1 [n = 9] from Type 2 VWD samples [phenotypes 2A and 2B; n = 11]. In summary, MAB-based systems can be used to measure VWF and confirm a diagnosis of VWD, as well as exhibiting some VWD-subtype-discriminatory capabilities. However, better evidence of VWF-discordance was usually achieved using the VWF:RCof (agglutination) assay, while the greatest degree of VWFdiscordance was consistently observed using the VWF:CBA assay. In conclusion, the VWF:CBA assay proved to offer the best diagnostic predictive tool for a Type 2 VWD defect, while MAB-based systems appear to be less effective in this regard. Abbreviations: HMW: High Molecular Weight [VWF], MAB: Monoclonal antibody (/antibodies), PNP: Pooled Normal Plasma, RIPA: Ristocetin induced platelet aggregation, VWD: von Willebrands disease (/disorder), VWF: von Willebrand Factor, VWF:Ag: von Willebrand Factor Antigen (assay), VWF:CBA: Collagen Binding [Activity] Assay for VWF, VWF:RCof: Ristocetin Cofactor Assay for VWF

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Perréard, Marion, Vianney Bastit, Lucie Lecouflet, Guillaume Desmartin, Romane Florent, Corinne Jeanne, Juliette Thariat, Emmanuel Babin, Laurent Poulain et Louis-Bastien Weiswald. « Abstract 222 : Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments ». Cancer Research 84, n^o 6_Supplement (22 mars 2024) : 222. http://dx.doi.org/10.1158/1538-7445.am2024-222.

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Abstract The risk of relapse or recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is high despite the combination of surgery and radiochemotherapy, responsible for high toxicity. It is crucial to develop new therapeutic strategies and to identify patients likely to benefit from these treatments. Patient-Derived Tumor Organoids (PDTO) are three-dimensional multicellular structures derived from patient tumor samples and faithfully reproduce the histological and molecular characteristics of the original tumor. A growing body of research indicates that PDTO may predict the clinical response, representing a major opportunity for the development of new therapeutic strategies and precision medicine. The purpose of this translational study is to generate PDTO from HNSCC patients to evaluate their predictive value. PDTO were obtained after dissociation of tumor specimen of HNSCC patients through the setup of the clinical study ORGAVADS (NCT04261192). Tumor cells were embedded in extracellular matrix and cultured in specific medium. Histological and immunohistochemical characterizations were performed to validate the resemblance between PDTO and their original tumor. Response of PDTO to chemotherapy, radiotherapy and innovative therapies were evaluated by live-cell imaging and viability assays. The culture conditions were optimized to improve the success rate of PDTO establishment which now exceeded 50%. Twenty-one PDTO lines have been established and showed histological characteristics close to the original tumor. Expression of immunohistochemical tumor markers p53, p40, p63, and p16 were similar in tumors and paired PDTO. Functional assays were performed on 15 PDTO to analyze the response to treatments (cisplatin, olaparib, X-Rays) and heterogeneity of response was observed between the models. When clinical response was available, PDTO derived from good responders showed high sensitivity to treatments while PDTO from bad responders showed low sensitivity to treatments. Interestingly, two PDTO lines derived from HPV+ oropharyngeal tumors displayed very high sensitivity to cisplatin, matching with the patient's profile and response. Our results showed feasibility to derive PDTO from HNSCC and to perform functional assay to assess their response to different treatments. The first studies of correlation between PDTO and patient responses are promising and will be further investigated in more patients. This will allow to demonstrate the predictive value of PDTO from HNSCC with the purpose to develop a clinically compatible predictive functional assay. Citation Format: Marion Perréard, Vianney Bastit, Lucie Lecouflet, Guillaume Desmartin, Romane Florent, Corinne Jeanne, Juliette Thariat, Emmanuel Babin, Laurent Poulain, Louis-Bastien Weiswald. Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 222.

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Parmar, Harsh V., Nitya Chakraborty et Upendra P. Hegde. « Peripheral blood T-cell functional assay as a predictive biomarker of immune-therapy of metastatic melanoma. » Journal of Clinical Oncology 36, n^o 15_suppl (20 mai 2018) : e21593-e21593. http://dx.doi.org/10.1200/jco.2018.36.15_suppl.e21593.

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Sovadinová, Iva, Brad L. Upham, James E. Trosko et Pavel Babica. « Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing ». International Journal of Molecular Sciences 22, n^o 16 (20 août 2021) : 8977. http://dx.doi.org/10.3390/ijms22168977.

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Dysregulation of gap junction intercellular communication (GJIC) is recognized as one of the key hallmarks for identifying non-genotoxic carcinogens (NGTxC). Currently, there is a demand for in vitro assays addressing the gap junction hallmark, which would have the potential to eventually become an integral part of an integrated approach to the testing and assessment (IATA) of NGTxC. The scrape loading-dye transfer (SL-DT) technique is a simple assay for the functional evaluation of GJIC in various in vitro cultured mammalian cells and represents an interesting candidate assay. Out of the various techniques for evaluating GJIC, the SL-DT assay has been used frequently to assess the effects of various chemicals on GJIC in toxicological and tumor promotion research. In this review, we systematically searched the existing literature to gather papers assessing GJIC using the SL-DT assay in a rat liver epithelial cell line, WB-F344, after treating with chemicals, especially environmental and food toxicants, drugs, reproductive-, cardio- and neuro-toxicants and chemical tumor promoters. We discuss findings derived from the SL-DT assay with the known knowledge about the tumor-promoting activity and carcinogenicity of the assessed chemicals to evaluate the predictive capacity of the SL-DT assay in terms of its sensitivity, specificity and accuracy for identifying carcinogens. These data represent important information with respect to the applicability of the SL-DT assay for the testing of NGTxC within the IATA framework.

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Donnelly, Liam L., Tyler C. Hogan, Sean M. Lenahan, Gopika Nandagopal, Jenna G. Eaton, Meagan A. Lebeau, Cai L. McCann et al. « Functional assessment of somatic STK11 variants identified in primary human non-small cell lung cancers ». Carcinogenesis 42, n^o 12 (19 novembre 2021) : 1428–38. http://dx.doi.org/10.1093/carcin/bgab104.

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Abstract Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

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Capala, Marta E., Katrin S. Pachler, Iris Lauwers, Maarten A. de Korte, Nicole S. Verkaik, Hetty Mast, Brend P. Jonker et al. « Ex Vivo Functional Assay for Evaluating Treatment Response in Tumor Tissue of Head and Neck Squamous Cell Carcinoma ». Cancers 15, n^o 2 (12 janvier 2023) : 478. http://dx.doi.org/10.3390/cancers15020478.

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Background: Head and neck squamous cell carcinoma (HNSCC) displays a large heterogeneity in treatment response, and consequently in patient prognosis. Despite extensive efforts, no clinically validated model is available to predict tumor response. Here we describe a functional test for predicting tumor response to radiation and chemotherapy on the level of the individual patient. Methods: Resection material of 17 primary HNSCC patients was cultured ex vivo, irradiated or cisplatin-treated, after which the effect on tumor cell vitality was analyzed several days after treatment. Results: Ionizing radiation (IR) affected tumor cell growth and viability with a clear dose-response relationship, and marked heterogeneity between tumors was observed. After a single dose of 5Gy, proliferation in IR-sensitive tumors dropped below 30% of the untreated level, while IR-resistant tumors maintained at least 60% of proliferation. IR-sensitive tumors showed on average a twofold increase in apoptosis, as well as an increased number and size of DNA damage foci after treatment. No differences in the homologous recombination (HR) proficiency between IR-sensitive and –resistant tumors were detected. Cisplatin caused a decrease in proliferation, as well as induction of apoptosis, again with marked variation between the samples. Conclusions: Our functional ex vivo assay discriminated between IR-sensitive and IR-resistant HNSCC tumors, and may also be suitable for predicting response to cisplatin. Its predictive value is currently under investigation in a prospective clinical study.

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Day, William A., Suzanne L. Rasmussen, Beth M. Carpenter, Scott N. Peterson et Arthur M. Friedlander. « Microarray Analysis of Transposon Insertion Mutations in Bacillus anthracis : Global Identification of Genes Required for Sporulation and Germination ». Journal of Bacteriology 189, n^o 8 (2 février 2007) : 3296–301. http://dx.doi.org/10.1128/jb.01860-06.

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ABSTRACT A transposon site hybridization (TraSH) assay was developed for functional analysis of the Bacillus anthracis genome using a mini-Tn10 transposon which permitted analysis of 82% of this pathogen's genes. The system, used to identify genes required for generation of infectious anthrax spores, spore germination, and optimal growth on rich medium, was predictive of the contributions of two conserved hypothetical genes for the phenotypes examined.

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Grundy, Martin, Jones Thomas, Liban Elmi, Michael Hall, Nigel H. Russell et Monica Pallis. « Comparison of Two Rapid Predictive Methods for Therapeutic Targeting in Acute Myeloid Leukaemia Cells ». Blood 128, n^o 22 (2 décembre 2016) : 1704. http://dx.doi.org/10.1182/blood.v128.22.1704.1704.

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Abstract Background. AML blasts from different patients vary in the agents to which they are most responsive. With a plethora of novel agents to evaluate, there is a lack of predictive biomarkers to precisely assign targeted therapies to individual patients. Primary AML cells often survive poorly in vitro, thus confounding conventional cytotoxicity assays. Dynamic profiling, i.e. functional biomarker assays of responsiveness to chemotherapeutic agents, is an alternative approach to help further the move towards personalised therapy. The purpose of this work was to assess the potential of two same-day dynamic profiling assays in AML cell lines to predict response to chemotherapy. (i) Ribosomal protein S6 (rpS6) is a downstream substrate of PI3K/akt/mTOR/ p70S6 kinase and MAPK/p90S6 kinase pathways and is also dephosphorylated following DNA double strand breaks. It thus has the potential to function as a biomarker of responsiveness to several therapeutic agents. (ii) Cellular propensity for apoptosis can be interrogated via a functional assay termed BH3 profiling. Dynamic BH3 profiling can be used to predict cellular responses to therapy based on priming mitochondria with BH3 domain peptides and thus allowing early detection of pro-apoptotic drug effects on mitochondrial outer membrane permeabilisation. Methods.We measured effects of a short (four hour) incubation with drugs of interest on rpS6 phosphorylation and BH3 peptide-accelerated cytochrome C release by flow cytometry. Baseline rpS6 phosphorylation was determined by culture with the mTOR inhibitor Rapamycin and the MEK inhibitor U0126. BH3 profiling included permeabilisation with digitonin followed by mitochondrial exposure to PUMA BH3-derived peptide. To establish specificity for sensitive cells we also measured dose responses to the drugs in 12 diverse AML cell lines after 48 hours' culture. Results. RpS6 dephosphorylation at four hours closely predicted the 48 hour IC50 for FLT3 inhibitors, an hsp90 inhibitor and topoisomerase II inhibitors. ROC (predictive test) analysis of small molecule inhibitors showed that the assay was highly sensitive and specific with area under the curve (AUC) values of 1.0 (sorafenib), 1.0 (AC220) and 1.0 (17-AAG). RpS6 phosphorylation also predicted response to the double strand break inducing drugs, with AUC values of 1.0 (mylotarg), 0.83 (etoposide) and 0.82 (vosaroxin). In contrast, responses to cytarabine and ABT-199, likely independent of mTOR, MAPK or double strand break response pathways, are not predicted by rpS6 dephosphorylation. PUMA-BH3 peptide-induced cytochrome C release also closely predicted the 48 hour IC50 with AUC values for FLT3 inhibitors of 1.0 (sorafenib) and 1.0 (AC220), double strand break inducing drugs 1.0 (mylotarg), 1.0 (etoposide) and 1.0 (vosaroxin) and a Bcl-2 targeting agent 0.875 (ABT-199). Response to 17-AAG and cytarabine were not predicted by this assay. Preliminary analysis shows that differential responses within primary AML sample subsets can be interrogated with these methodologies. Conclusions. In conclusion, we have established that rpS6 dephosphorylation and/or PUMA-BH3 peptide-induced cytochrome C release predict chemoresponsiveness to tyrosine kinase inhibitors, topoisomerase II inhibitors, an hsp90 inhibitor and a Bcl-2 targeting agent in AML cell lines after short term culture. Both assays are sensitive, specific and amenable to leukaemic sub-population analysis and are thus suitable assays for further development towards use in a clinical setting. pRS6 can be performed on smaller samples and is less technically challenging than priming with PUMA-BH3 peptide, but does not take into account apoptosis resistance that may occur independently of pathways effecting pRS6 inhibition. Disclosures No relevant conflicts of interest to declare.

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Wilson, David H., David W. Hanlon, Gail K. Provuncher, Lei Chang, Linan Song, Purvish P. Patel, Evan P. Ferrell et al. « Fifth-Generation Digital Immunoassay for Prostate-Specific Antigen by Single Molecule Array Technology ». Clinical Chemistry 57, n^o 12 (1 décembre 2011) : 1712–21. http://dx.doi.org/10.1373/clinchem.2011.169540.

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BACKGROUND Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays. METHODS We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. RESULTS The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. CONCLUSIONS The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

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Hernández, Pilar, Julián Gorrochategui, Daniel Primo, Alicia Robles, José Luis Rojas, Ana Belén Espinosa, Cristina Gómez, Joaquín Martínez-López, Teresa A. Bennett et Joan Ballesteros. « Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry ». SLAS TECHNOLOGY : Translating Life Sciences Innovation 22, n^o 3 (24 mars 2017) : 325–37. http://dx.doi.org/10.1177/2472630317700346.

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Functional ex vivo assays that predict a patient’s clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients’ response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient’s sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.

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Klinker, Matthew W., Ross A. Marklein, Jessica L. Lo Surdo, Cheng-Hong Wei et Steven R. Bauer. « Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity ». Proceedings of the National Academy of Sciences 114, n^o 13 (10 mars 2017) : E2598—E2607. http://dx.doi.org/10.1073/pnas.1617933114.

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Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ–enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.

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McAleer, C. W., A. S. T. Smith, S. Najjar, K. Pirozzi, C. J. Long et J. J. Hickman. « Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system ». Journal of Applied Physiology 117, n^o 11 (1 décembre 2014) : 1398–405. http://dx.doi.org/10.1152/japplphysiol.00612.2014.

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The ability to accurately measure skeletal muscle functional performance at the single-cell level would be advantageous for exercise physiology studies and disease modeling applications. To that end, this study characterizes the functional response of individual skeletal muscle myotubes derived from adult rodent tissue to creatine treatment and chronic exercise. The observed improvements to functional performance in response to these treatments appear to correlate with alterations in hypertrophic and mitochondrial biogenesis pathways, supporting previously published in vivo and in vitro data, which highlights the role of these pathways in augmenting skeletal muscle output. The developed system represents a multiplexed functional in vitro assay capable of long-term assessment of contractile cellular outputs in real-time that is compatible with concomitant molecular biology analysis. Adoption of this system in drug toxicity and efficacy studies would improve understanding of compound activity on physical cellular outputs and provide more streamlined and predictive data for future preclinical analyses.

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Kristof, Jessica, Kellen Sakrison, Xiaoping Jin, Kenji Nakamaru, Matthias Schneider, Robert A. Beckman, Daniel Freeman, Cindy Spittle et Wenqin Feng. « Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non–Small Cell Lung Cancer Tissue ». Biomarker Insights 12 (1 janvier 2017) : 117727191769985. http://dx.doi.org/10.1177/1177271917699850.

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In preclinical studies, heregulin ( HRG) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti–epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non–small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG-positive and HRG-negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8, and EIF2B1), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

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Mazard, Thibault, Laure Cayrefourcq, Françoise Perriard, Hélène Senellart, Benjamin Linot, Christelle de la Fouchardière, Eric Terrebonne et al. « Clinical Relevance of Viable Circulating Tumor Cells in Patients with Metastatic Colorectal Cancer : The COLOSPOT Prospective Study ». Cancers 13, n^o 12 (13 juin 2021) : 2966. http://dx.doi.org/10.3390/cancers13122966.

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Background: Circulating tumor cells (CTCs) allow the real-time monitoring of tumor course and treatment response. This prospective multicenter study evaluates and compares the early predictive value of CTC enumeration with EPISPOT, a functional assay that detects only viable CTCs, and with the CellSearch® system in patients with metastatic colorectal cancer (mCRC). Methods: Treatment-naive patients with mCRC and measurable disease (RECIST criteria 1.1) received FOLFIRI–bevacizumab until progression or unacceptable toxicity. CTCs in peripheral blood were enumerated at D0, D14, D28, D42, and D56 (EPISPOT assay) and at D0 and D28 (CellSearch® system). Progression-free survival (PFS) and overall survival (OS) were assessed with the Kaplan–Meier method and log-rank test. Results: With the EPISPOT assay, at least 1 viable CTC was detected in 21% (D0), 15% (D14), 12% (D28), 10% (D42), and 12% (D56) of 155 patients. PFS and OS were shorter in patients who remained positive, with viable CTCs between D0 and D28 compared with the other patients (PFS = 7.36 vs. 9.43 months, p = 0.0161 and OS = 25.99 vs. 13.83 months, p = 0.0178). The prognostic and predictive values of ≥3 CTCs (CellSearch® system) were confirmed. Conclusions: CTC detection at D28 and the D0–D28 CTC dynamics evaluated with the EPISPOT assay were associated with outcomes and may predict response to treatment.

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Guzman, Grace, Sahana Bettadapura, William Jeremy Shelton-Correa, Taylor Brooks, Melissa Rayner, Christopher Wardell et Analiz Rodriguez. « Abstract 953 : Integration of functional precision medicine assay for high grade glioma management : A single institution experience ». Cancer Research 84, n^o 6_Supplement (22 mars 2024) : 953. http://dx.doi.org/10.1158/1538-7445.am2024-953.

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Abstract Despite the advances in precision oncology, genomic approaches have not significantly improved outcomes for high grade gliomas (HGGs) as once was anticipated. Recurrent HGG pose even greater therapeutic challenge. The integration of functional precision medicine (FPM), in which drugs screens are performed on live tissue, into clinical routine practice presents a promising alternative by offering personalized treatments based on the unique characteristics of the patient’s tumor. Here, we present our institutional experience of integrating a spheroid-based drug screening assay (3D PredictTM Glioma) in the treatment management of HGG patients to evaluate its feasibility and efficacy as a therapeutic tool in a clinical setting. We also sought to determine factors that related to assay success. Tissue from intracranial lesions of patients with presumed HGG and planned surgical resection at our institution were collected for ex vivo 3D cell culture and challenged with 12 drug agents to determine patient-specific response parameters. The impact of potential variables, such as ki67, tumor pathology, and shipment timing, on the assay’s success was evaluated. Clinical correlation was established between ex vivo response and clinical response in HGG patients. 57 samples, including 24 upfront HGGs, 32 recurrent HGGs, and one medulloblastoma, were sent for spheroid formation and drug testing. In total, 57.9% (33/57) or 23% (13/57) of the assays were successful and resulted in complete or partial functional profiling data, respectively. We conducted a multivariable analysis of our cohort to determine which variables (i.e. tumor volume sent, ki-67 proliferation index, tumor percentage determined by DNA sequencing by Tempus Lab, recurrent status, sample shipment time) were predictive of assay success. The only variable significantly associated with assay success was tumor percentage with samples comprised of >70% tumor leading to assay success, 40%-70% leading to 60% chance of success, and <40% having 0% success. Among 46 patients with partial or complete assay success, 26 patients (56.5%) had a targetable mutation and 17/26 yielded a moderate or full response to one or more of their corresponding targeted drugs in the assay. Treatments were newly administered or altered from prior treatments in 40/57 patients (70%) based on FPM results. Our study demonstrates the technical feasibility of incorporating FPM approaches in the development of treatment regimens for patients with HGG. Success was correlated to tumor percentage present in the sample which indicates intraoperative communication between surgeon and pathologist can lead to improved assay results. Our institutional protocol demonstrates that Integrating FPM into routine clinical practice is possible and can serve as a valuable guide in the refinement of therapeutic recommendations for HGG patients. Citation Format: Grace Guzman, Sahana Bettadapura, William Jeremy Shelton-Correa, Taylor Brooks, Melissa Rayner, Christopher Wardell, Analiz Rodriguez. Integration of functional precision medicine assay for high grade glioma management: A single institution experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 953.

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Wong, Edward C. C., Laura A. Worfolk, Caixia Bi, Lina J. Noh, Andrew Espinoza, William W. Wu, Mervyn A. Sahud, Frederick K. Racke et Jeffrey S. Dlott. « Heparin-Induced Thrombocytopenia Testing : ELISA Based Anti-Platelet Factor 4 Polyspecific and IgG-Specific Antibody Detection Assays Compared to the Unfractionated Heparin Serotonin Release Assay ». Blood 138, Supplement 1 (5 novembre 2021) : 3209. http://dx.doi.org/10.1182/blood-2021-145373.

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Abstract Introduction: Antibodies that cause heparin-induced thrombocytopenia (HIT) can be detected with either antigenic or functional assays. Previously, it has been shown that antigenic (ELISA based) assays that detect anti-platelet factor 4 (anti-PF4) IgG, IgM, or IgA (polyspecific) antibodies are more sensitive but less specific than functional assays such as the unfractionated serotonin release assay (UFH SRA), and that the use of anti-PF4 assays that detect IgG antibodies only, would increase the specificity but decrease the sensitivity of these assays for the detection of HIT antibodies that are prothrombotic (associated with positive functional assay). To date large epidemiologic studies have not confirm these findings. To evaluate the relative performance of anti-PF4 polyspecific and IgG-specific antibodies in their ability to detect prothrombotic HIT antibodies, we evaluated results of non-reflexive HIT panels that contained either anti-PF4 polyspecific or IgG-specific assays and unfractionated heparin serotonin release assays over an eight-year period at a U.S. reference laboratory. Methods: Test results for 2 HIT detection panels were compared: 1 panel had UFH SRA plus the polyspecific PF4 ENHANCED® assay (GTI Diagnostics, Waukesha, WI) and 1 panel had UFH SRA plus the IgG-specific Zymutest HIA IgG assay (Hyphen Biomed, France). Test results were from the last 4 years of use for each panel (2009 to 2012 for the polyspecific panel; 2017 to 2020 for the IgG-specific panel). UFH SRA was performed as described by Sheridan et al, (1986) with positivity defined as ≥20% serotonin release by low dose UFH and >50% suppression of release at high dose (100 U/mL) UFH. For each year and assay, test results were stratified by optical density (OD) results, and the percent of results positive by UFH SRA was determined for each OD range. Median yearly UFH SRA positivity rates for each OD interval were compared for anti-PF4 polyspecific vs IgG-specific antibody assays using non-parametric statistical testing, Mann-Whitney U test, two-tailed, with significance defined as <0.05. Results: HIT panels with either ELISA based assays detecting either anti-PF4 polyspecific or IgG specific antibodies demonstrated increasing UFH SRA positivity rates as OD increased. Approximately 50% UFH SRA positivity occurred when OD was in the 2.000 to range. No significant differences in SRA positivity were seen at any positive OD interval when comparing anti-PF4 polyspecific vs IgG-specific assays. A small but significant difference was seen when OD results were considered This observation may have been due to a in the review process (2017-2020): when a UFH SRA result was positive with a negative OD result, repeat UFH SRA testing was performed. Conclusions: Our study demonstrates that the correlations of UFH SRA positivity and OD measurements are similar for anti-PF4 IgG-specific and polyspecific antibody assays. These results suggest the assay types may perform similarly for the detection of HIT and importantly provide important predictive information as to when an optical density value will lead to a positive UFH SRA result. Figure 1 Figure 1. Disclosures Wong: Quest Diagnostics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Worfolk: Quest Diagnostics: Current Employment. Bi: Quest Diagnostics: Current Employment. Noh: Quest Diagnostics: Current Employment. Espinoza: Quest Diagnostics: Current Employment. Wu: Quest Diagnostics: Current Employment. Sahud: Quest Diagnostics: Current Employment. Racke: Quest Diagnostics: Current Employment. Dlott: Quest Diagnostics: Current Employment.

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Bacot, Silvia M., Taylor A. Harper, Rebecca L. Matthews, Christie Jane Fennell, Adovi Akue, Mark A. KuKuruga, Shiowjen Lee, Tao Wang et Gerald M. Feldman. « Exploring the Potential Use of a PBMC-Based Functional Assay to Identify Predictive Biomarkers for Anti-PD-1 Immunotherapy ». International Journal of Molecular Sciences 21, n^o 23 (27 novembre 2020) : 9023. http://dx.doi.org/10.3390/ijms21239023.

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The absence of reliable, robust, and non-invasive biomarkers for anti- Programmed cell death protein 1 (PD-1) immunotherapy is an urgent unmet medical need for the treatment of cancer patients. No predictive biomarkers have been established based on the direct assessment of T cell functions, the primary mechanism of action of anti-PD-1 therapy. In this study, we established a model system to test T cell functions modulated by Nivolumab using anti-CD3 monoclonal antibody (mAb)-stimulated peripheral blood mononuclear cells (PBMCs), and characterized T cell functions primarily based on the knowledge gained from retrospective observations of patients treated with anti-PD-1 immunotherapy. During a comprehensive cytokine profile assessment to identify potential biomarkers, we found that Nivolumab increases expression of T helper type 1 (Th1) associated cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a subset of donors. Furthermore, Nivolumab increases production of Th2, Th9, and Th17 associated cytokines, as well as many proinflammatory cytokines such as IL-6 in a subset of donors. Conversely, Nivolumab treatment has no impact on T cell proliferation, expression of CD25, CD69, or Granzyme B, and only modestly increases in the expansion of regulatory T cells. Our results suggest that assessment of cytokine production using a simple PBMC-based T cell functional assay could be used as a potential predictive marker for anti-PD-1 immunotherapy.

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Hinsberger, Manuel, Julia Becker-Kettern, Wiebke M. Jürgens-Wemheuer, Joachim Oertel et Walter J. Schulz-Schaeffer. « Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of ARID1A in Tissue Lysates ». Cancers 15, n^o 16 (14 août 2023) : 4096. http://dx.doi.org/10.3390/cancers15164096.

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ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a ‘goodness of fit’ of R2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value.

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Eckert, A., T. Mikoteit, J. Beck, U. M. Hemmeter, S. Brand, K. Schmitt, R. Bischof, A. Delini-Stula et E. Holsboer-Trachsler. « Assessment of mature serum brain-derived neurotrophic factor (BDNF) is not superior to total serum BDNF in prediction of antidepressant treatment outcome ». European Psychiatry 33, S1 (mars 2016) : S410. http://dx.doi.org/10.1016/j.eurpsy.2016.01.1480.

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BackgroundSerum BDNF levels are decreased in major depressive disorder (MDD) and tend to normalize under antidepressant treatment, serving as a treatment outcome predictor. BDNF is initially synthetized as precursor protein proBDNF and is cleaved to mature BDNF (mBDNF) while only the latter exerts neurotrophic activity.AimThe aim was to explore if a specific enzyme-linked immunosorbent assay (ELISA) kit for mBDNF in serum would be superior to the unspecific assessment of total serum BDNF in predicting treatment response in MDD.MethodsTwenty-five patients with MDD underwent standardized treatment with duloxetine. Severity of depression was measured by Hamilton Depression Rating Scale (HDRS) at baseline (BL), after one (W1), two (W2) and six weeks (W6) of treatment. Treatment response was defined as a HDRS ≥ 50% reduction of BL score at W6. mBDNF and total BDNF serum levels were determined at BL, W1 and W2.ResultsA high and stable correlation was found between mBDNF and total BDNF serum levels over all measurements. The predictive value of mBDNF BL levels and mBDNFΔW1 to response was similar to that of total BDNF BL and total BDNFΔW1. The assessment of serum mBDNF was not superior to total BDNF in prediction of treatment outcome.ConclusionsNot only baseline total BDNF but also mBDNF is predictive to treatment outcome. The later might represent the main player in this respect, which supports the idea of a functional link between neuroplasticity and MDD.Disclosure of interestThe authors have not supplied their declaration of competing interest.

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Hurton, Lenka, R. Iram Siddik, Harjeet Singh, Simon Olivares, Brian Rabinovich, William Hildebrand, Dean Lee et al. « Identifying NK-Cell Donors for Cell Therapy Based on Functional Phenotype. » Blood 110, n^o 11 (16 novembre 2007) : 3271. http://dx.doi.org/10.1182/blood.v110.11.3271.3271.

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Abstract Donor natural killer (NK) cells after haploidentical hematopoietic stem-cell transplantation (HSCT) and infusion of haploidentical NK-cells have demonstrated a therapeutic effect. NK alloreactivity resulting from appropriate Killer cell Ig-like receptor (KIR)-ligand disparity in human-leukocyte-antigen (HLA)-haplotype mismatched HSCT has resulted in improved engraftment and decreased incidence of leukemia relapse. Yet, not all patient-donor pairs benefit for an allogeneic NK-cell effect. To identify NK-cell donors with a suitable KIR-ligand mismatch, we have developed a functional assay to measure NK-cell killing through KIR-ligand interactions. NK-cell lysis of target cells is blocked by inhibitory KIR that recognize classical HLA class I allotypes and HLA mismatches of an altered allelic repertoire, as in haploidentical HSCT, leading to KIR-ligand mismatch and alloreactive NK cell-mediated target killing (Figure 1A). A cytotoxicity assay was developed based on the NK-cell target HLAnull 721.221 cells, and a panel of targets with enforced expression of HLA genes recognized by KIR. After the killing assay was optimized for high throughput and sensitivity, we used the panel of targets to determine whether bulk populations of donor NK cells could be predicted to kill based on KIR and HLA typing. The results demonstrate patterns of target-cell lysis for the KIR repertoires corresponding, for some donors, with predicted donor-versus-recipient NK-cell alloreactivity (Figure 1B). A relative inhibition of HLA+ target-cell lysis of >30% was associated with binding of KIR to introduced HLA class I molecules. The benefit of this assay to transplant physicians is a tool to actually measure phenotype (lysis), rather than relying on predictive models based on genotype. This assay will be combined with typing data to help identify donors with NK-cell killing function for recipients of haploidentical HSCT and infusion of haploidentical NK cells. Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box). Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box).

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DeBois, William J., Junli Liu, Leonard Y. Lee, Leonard N. Girardi, Charles Mack, Anthony Tortolani, Karl H. Krieger et O. Wayne Isom. « Diagnosis and treatment of heparin-induced thrombocytopenia ». Perfusion 18, n^o 1 (janvier 2003) : 47–53. http://dx.doi.org/10.1191/0267659103pf637oa.

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Heparin-induced thrombocytopenia (HIT) is a major side effect secondary to the administration of heparin. This syndrome is serious and potentially life threatening. This response is the result of antibodies formed against the platelet factor 4 (PF4)/heparin complex. The incidence of this immune-mediated syndrome has been estimated to be 1-3% of all patients receiving heparin therapy. The occurrence of HIT in patients requiring full anticoagulation for cardiopulmonary bypass (CPB), therefore, presents a serious challenge to the cardiac surgery team. The diagnosis of HIT should be based on both clinical and laboratory evidence. While functional assays, platelet aggregation tests, and the serotonin release assay can be used to support the diagnosis, the negative predictive value of these tests is generally less than 50%. In contrast, although non-functional antibody detection assays are more sensitive, they have a low specificity. HIT can be treated in several ways, including cessation of all heparin and giving an alternative thrombin inhibitor, platelet inhibition followed by heparin infusion, and the use of low molecular weight heparins. In this presentation, the pathology and current diagnostic tests, as well as the successful management of patients with HIT undergoing CPB at New York Presbyterian Hospital, are reviewed.

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Goldstein, Richard A. « Skeletal Muscle Injury Biomarkers : Assay Qualification Efforts and Translation to the Clinic ». Toxicologic Pathology 45, n^o 7 (octobre 2017) : 943–51. http://dx.doi.org/10.1177/0192623317738927.

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Skeletal muscle (SKM) injury or myopathy results in structural or functional defects in SKMs that can be caused by variety of factors such as (1) genetic, (2) drug-induced, (3) disease progression (cachexia), or (4) aging (sarcopenia). Creatine kinase (CK) and aspartate transaminase (AST) activity assays have been routinely used as SKM injury biomarkers, but they lack sensitivity and tissue specificity. In collaboration with the Predictive Safety Testing Consortium, we evaluated the diagnostic performance of a muscle injury biomarker panel (MIP) compared to CK and AST and their correlation with the histology scores across 34 different rat studies. The MIP panel included the analytes skeletal troponin I, myosin light chain 3, fatty acid binding protein 3, and a CK mass (versus activity) assay. The area under the receiver operator characteristic curve for MIP panel ranged from 0.82 to 0.91 as compared to 0.71 and 0.82 for CK and AST activity assays, respectively. Because the MIP biomarkers outperformed the routine biomarkers, the European Medicines Agency and U.S. Food and Drug Administration posted Letters of Support encouraging further study of these analytes and acknowledged the utility of the MIP panel. Ongoing efforts are directed toward the application of the MIP panel biomarkers in clinical studies and regulatory qualification.

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Mahamed, Deeqa, Michael Cohen, Stephen Li, Lauren Tracey, Huihui Yao, Christina Loh et Leslie Fung. « Abstract 6671 : Simplifying high-parameter phenotypic and functional characterization of cancer immune cells ». Cancer Research 83, n^o 7_Supplement (4 avril 2023) : 6671. http://dx.doi.org/10.1158/1538-7445.am2023-6671.

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Abstract Interrogating immune cell composition and function in patients with cancer is critical for making disease prognoses, monitoring clinical efficacy of tumor immunotherapies, identifying novel therapeutic targets, and discovering predictive biomarkers of disease. Both the adaptive and innate arms of the immune system play important roles in generating pro- or anti-tumor milieus. In particular, natural killer (NK) cells are gaining increasing attention as potential cell-based therapies in immuno-oncology, including in adoptive transfer of chimeric antigen receptor (CAR) and iPSC-derived NK cells or off-the-shelf NK cell lines to target multiple myeloma. Since NK cells can also indirectly impact CAR T cell or antibody-based immunotherapies, characterizing these cells using optimized and reproducible assays is critical. CyTOF® is a high-plex flow cytometry technology that exploits metal-isotope-tagged antibodies to probe cellular phenotypes and functions. In contrast to fluorescence-based conventional and spectral flow cytometry, CyTOF experimental workflows are streamlined as autofluorescence is not an issue and signal spillover is minimal, allowing rapid design and application of 40-plus-marker panels. To expand on the increasing clinical and preclinical utility of the 30-marker Maxpar® Direct™ Immune Profiling Assay™ (Maxpar Direct Assay), we developed nine add-on Expansion Panels for deeper phenotyping of specific cell types and activation states, including panels designed to characterize ex vivo and activated myeloid cells, T cells, and NK cells. Peripheral blood mononuclear cells from healthy donors and donors with multiple myeloma were stimulated in vitro, then stained in the 30-antibody Maxpar Direct Assay tube with the NK Cell Expansion Panel (CD181, NKp30, NKp46, PD-1, NKG2A, ICOS, and TIGIT) or the T Cell Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4-1BB) as drop-in antibodies. Surface staining was followed by intracellular staining with the Basic Activation Expansion Panel antibodies (IL-2, TNFα, IFNγ, perforin, granzyme B) after cell fixation and permeabilization. Anti-CD107a was added during stimulation to measure degranulation. Samples were acquired on a CyTOF XT™ instrument in automated batch acquisition mode. Automated analysis was performed with Maxpar Pathsetter™ software to enumerate immune cell types and quantify marker expression. In addition to the 37 populations identified by Pathsetter with the base Maxpar Direct Assay, the new Expansion Panels allowed deeper profiling of NK and T cells, while the Basic Activation Panel revealed their cytokine responsiveness and cytotoxic potential. Thus, the optimized single-tube Maxpar Direct Assay is a powerful tool that can be further expanded and customized with predefined panels or antibodies to comprehensively study immune cells in health and disease. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Deeqa Mahamed, Michael Cohen, Stephen Li, Lauren Tracey, Huihui Yao, Christina Loh, Leslie Fung. Simplifying high-parameter phenotypic and functional characterization of cancer immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6671.

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Mayr, Nina A., William T. C. Yuh, David Jajoura, Jian Z. Wang, Simon S. Lo, Joseph F. Montebello, Kyle Porter, Dongqing Zhang, D. Scott McMeekin et John M. Buatti. « Ultra-early predictive assay for treatment failure using functional magnetic resonance imaging and clinical prognostic parameters in cervical cancer ». Cancer 116, n^o 4 (15 février 2010) : 903–12. http://dx.doi.org/10.1002/cncr.24822.

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Mirzaei, Mahsa, Irini Furxhi, Finbarr Murphy et Martin Mullins. « Employing Supervised Algorithms for the Prediction of Nanomaterial’s Antioxidant Efficiency ». International Journal of Molecular Sciences 24, n^o 3 (1 février 2023) : 2792. http://dx.doi.org/10.3390/ijms24032792.

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Reactive oxygen species (ROS) are compounds that readily transform into free radicals. Excessive exposure to ROS depletes antioxidant enzymes that protect cells, leading to oxidative stress and cellular damage. Nanomaterials (NMs) exhibit free radical scavenging efficiency representing a potential solution for oxidative stress-induced disorders. This study aims to demonstrate the application of machine learning (ML) algorithms for predicting the antioxidant efficiency of NMs. We manually compiled a comprehensive dataset based on a literature review of 62 in vitro studies. We extracted NMs’ physico-chemical (P-chem) properties, the NMs’ synthesis technique and various experimental conditions as input features to predict the antioxidant efficiency measured by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Following data pre-processing, various regression models were trained and validated. The random forest model showed the highest predictive performance reaching an R2 = 0.83. The attribute importance analysis revealed that the NM’s type, core-size and dosage are the most important attributes influencing the prediction. Our findings corroborate with those of the prior research landscape regarding the importance of P-chem characteristics. This study expands the application of ML in the nano-domain beyond safety-related outcomes by capturing the functional performance. Accordingly, this study has two objectives: (1) to develop a model to forecast the antioxidant efficiency of NMs to complement conventional in vitro assays and (2) to underline the lack of a comprehensive database and the scarcity of relevant data and/or data management practices in the nanotechnology field, especially with regards to functionality assessments.

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Sánchez-Espiridión, Beatriz, Carlos Montalbán, Ángel López, Javier Menárguez, Pilar Sabín, Carmen Ruiz-Marcellán, Andrés Lopez et al. « A molecular risk score based on 4 functional pathways for advanced classical Hodgkin lymphoma ». Blood 116, n^o 8 (26 août 2010) : e12-e17. http://dx.doi.org/10.1182/blood-2010-02-270009.

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Abstract Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P < .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

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Eppert, Kolja, Katsuto Takenaka, Björn Nilsson, Eric R. Lechman, Vicki Ling, Joseph Beyene, Angelo J. Canty et al. « Leukemic and Normal Stem Cell Transcriptional Signatures Determined by Functional Assays Are Predictive of the Overall Survival of AML Patients. » Blood 114, n^o 22 (20 novembre 2009) : 389. http://dx.doi.org/10.1182/blood.v114.22.389.389.

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Abstract Abstract 389 Normal hematopoiesis and acute myeloid leukemia (AML) are organized as hierarchies with stem cells, which possess extensive self-renewal and proliferative capacity, at the apex. Although there is definitive evidence from experimental models for the existence of leukemic stem cells (LSC) in some human leukemias, the relevance of LSC to human disease progression is still lacking. While chemotherapeutic treatment of AML patients typically results in disease remission, the majority of patients will eventually relapse and succumb to the disease, indicating that residual LSC are not eliminated by current treatment. We hypothesize that stem cell derived gene expression profiles may be more clinically relevant than those derived from examination of bulk leukemia samples. Here we show the clinical significance of novel stem cell related expression profiles derived from 25 functionally validated human leukemia stem cell populations and 6 normal hematopoietic stem cell populations. Little is currently known about the molecular regulatory networks that govern human LSC or hematopoietic stem cells (HSC). Therefore, we have carried out global mRNA gene expression profiling of FACS sorted subpopulations of cells enriched for human stem cells, progenitor cells and mature cells from 16 AML primary patient samples and 3 cord blood samples to investigate these pathways. Similar to normal hematopoietic stem cells, leukemia stem, progenitor and mature cells can be sorted using CD34 and CD38 markers. Due to the heterogeneous nature of AML, it is vital that quantitative functional assays are used to characterize the LSC and progenitor activity in each sorted fraction. In vitro cell suspension cultures and methylcellulose colony formation assays were performed to characterize progenitor and blast populations. Importantly, we applied a novel and improved in vivo SCID leukemia initiating cell assay to substantiate the presence of LSC activity in each sorted fraction of 16 AML patient samples. With this enhanced assay, LSC were detected in the expected CD34+/CD38- population. However, in the majority of AML samples, LSC were detected in at least one additional fraction, demonstrating the importance of functional validation when interpreting global gene expression profiles of sorted stem cell populations. LSC and HSC specific signatures were identified following a statistical analysis that compared fractions with stem cell activity against those without (25 LSC vs 29 non-LSC; 6 HSC vs 6 non-HSC). When applied to an independent gene expression data set from 160 cytogenetically normal AML samples, a 25 probe LSC signature was the strongest predictor of overall survival (p<0.0001, HR=2.6, 95%CI 1.8-4.0, median survival 236 vs 999 days; Figure 1a). Furthermore, the 225 probe HSC specific signature derived from normal cells also provided a strong predictor of survival (p<0.0001, HR=2.3, 95%CI 1.5-3.4, median survival 238 vs 741 days; Figure 1b). We queried the gene expression-based chemical genomic database Connectivity Map with the LSC-related gene list and found a negative correlation between the genes in the LSC profile and the expression of genes that are transcriptionally induced following treatment with common chemotherapeutic compounds such as doxorubicin, suggesting resistance to chemotherapy as one possible mechanism for the correlation of the stem cell signatures with survival. Together these data support the hypothesis that the biological determinants that underlie stemness in both normal and leukemic cells are predictors of poor outcome, and are potential targets for novel therapy. Disclosures: No relevant conflicts of interest to declare.

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Zhao, Tingting, Yuqin Zhou, Xiujuan Wu, Zaihui Peng, Pingping Gao, Na Sun, Yi Zhang et Xiaowei Qi. « Effect of transcription factor WT1 on triple-negative breast cancer metastasis through PFKFB4-mediated glycolysis. » Journal of Clinical Oncology 42, n^o 16_suppl (1 juin 2024) : e13145-e13145. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.e13145.

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e13145 Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high probability of metastasis as well as a lack of specific targets and targeted therapeutics. Preliminary study suggested that Wilms' tumor gene 1 (WT1) is highly expressed in breast cancer patients with poor prognosis and may promote TNBC metastasis, but the underlying mechanism remains poorly defined. Methods: WT1 expression was evaluated by immunohistochemistry (IHC) in breast cancer patients. Kaplan–Meier survival analysis was performed to assess the prognostic significance of WT1 expression. WT1 was silenced in MDA-MB-231 and BT549 cells or overexpressed in HCC1806 cells. qRT-PCR and Western blot were used to detect the WT1 expression in tissues and cells. Wound healing assays, transwell assays and 3D spheroid assays were used to examine the migration and invasion abilities in TNBC cells. The lung metastasis model of mice was used to evaluate metastasis of TNBC in vivo. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome sequencing (RNA-seq) were performed to find PFKFB4, a downstream target gene for WT1. ChIP-PCR and dual-luciferase reporter assays were used to explore the transcriptional regulation of PFKFB4 by WT1. Seahorse XF glycolysis stress assay, glucose uptake assay, and lactate production assay were used to investigate the role of WT1 in regulating glycolysis metabolites. Results: WT1 was highly expressed in TNBC and correlated to poor prognosis in TNBC patients. Functional assays revealed that WT1 promoted TNBC cell metastasis in vitro and in vivo. Our mechanistic investigations demonstrated that WT1 promoted TNBC cells migration and invasion by transcriptionally activating the expression of PFKFB4.This action leaded to increased glycolytic capacity, glucose uptake, and lactate production in cancer cells, therefore promoting metastasis of TNBC. Clinically, the combined expression of WT1 and PFKFB4 provides a reliable predictive biomarker for the prognosis of TNBC patients. Conclusions: Our findings reveal a molecular mechanism of WT1 promoting TNBC metastasis, which provides new targets for the precision treatment of TNBC and new perspectives for the development of targeted metabolic anticancer drugs.

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Sarić-Matutinović, Marija, Tanja Diana, Biljana Nedeljković-Beleslin, Jasmina Ćirić, Miloš Žarković, Iva Perović-Blagojević, George Kahaly et Svetlana Ignjatović. « Sensitivity of three thyrotropin receptor antibody assays in thyroid-associated orbitopathy ». Journal of Medical Biochemistry 41, n^o 2 (2022) : 211–20. http://dx.doi.org/10.5937/jomb0-34718.

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Background: Thyrotropin receptor autoantibodies (TSH-RAb) are indispensable biomarkers in the laboratory assessment of thyroid-associated orbitopathy (TAO). Clinical sensitivity of three different assays for TSH-R-Ab determination was evaluated in patients with TAO. Methods: 87 consecutive TAO patients were enrolled and their serum samples analyzed in parallel with three assays. An ECLIA competitive binding and a chemiluminescent bridge immunoassay were used to measure total and binding TSH-R-Ab concentration, while their functional activity was determined using a stimulatory TSH-R-Ab (TSAb) cellbased bioassay. Results: Compared to the two binding assays (ECLIA p<0.001, bridge p=0.003), the TSAb bioassay was more sensitive pertaining to the positive detection of TSH-R-Ab in TAO patients. No difference (p=0.057) was noted between the ECLIA and bridge assays regarding sensitivity rate. All patients with active and/or moderate-to-severe TAO tested positive in the TSAb bioassay (100% and 100%, respectively), while the positivity rates for bridge and ECLIA binding assays were 89.7% and 82.1% for active TAO, and 90.2% and 86.3% for severe TAO, respectively. Negative predictive values of the bioassay, bridge, and ECLIA assays were 100%, 75%, and 71%, respectively for active TAO, and 100%, 86%, and 71%, respectively for moderate-to-severe TAO. The superiority of the bioassay was most prominent in euthyroid (ET) TAO. Positivity rates of the TSAb bioassay, bridge and ECLIA binding assays were 89.6%, 75%, and 64.6%, respectively for inactive TAO; 86.1%, 69.4%, and 52.8%, respectively for mild TAO; 87.5%, 62.5%, and 12.5%, respectively for euthyroid TAO. The bridge assay correlated better with the ECLIA binding assay (r=0.893, p<0.001), compared to the bioassay (r=0.669, p<0.001). Conclusions: In patients with TAO of various activity and severity, the TSAb bioassay demonstrates a superior clinical performance compared to both ECLIA and bridge binding assays.

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Kwok, Jaime, Ming Yang, John K. Wu, Cedric J. Carter et Shannon Jackson. « High False Positive Rate Of An ELISA Screen For The Detection Of Anti-Factor VIII Antibodies In Congenital Hemophilia A ». Blood 122, n^o 21 (15 novembre 2013) : 3586. http://dx.doi.org/10.1182/blood.v122.21.3586.3586.

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Abstract Introduction Anti-factor VIII antibodies are a serious complication of factor replacement therapy in hemophilia A patients. Neutralizing antibodies could previously only be detected via the functional and gold-standard Bethesda assay with or without the Nijmegen modification. The introduction of an enzyme-linked immunosorbent assay (ELISA) screening test provides a less laborious alternative to the Bethesda assay and also has a high sensitivity. Unlike the Bethesda assay, the ELISA can potentially detect non-neutralizing antibodies, which raise the possibility of false positive screens. As it is important that both clinicians and the coagulation laboratory understand the clinical performance of ELISA screening, this study evaluated the sensitivity and specificity of ELISA and Bethesda assays used in both a laboratory validation study and in subsequent clinical experience. Methods Results from all active adult and pediatric congenital hemophilia A patients who underwent both Bethesda assay and ELISA in British Columbia, Canada as of July 2013 were included in this study. The sensitivity and specificity were compared to the provincial coagulation laboratory validation study of the GTI Factor 8 Antibody ELISA kit against the classical Bethesda assay conducted in both hemophilia A and normal controls in 2010. Optical density (OD) readings were retrieved for eligible samples and from the laboratory validation study. Results 35 samples from 26 different patients were used in the validation study performed in 2010. Of 147 adult and 86 pediatric hemophilia A clinic patients active during the study period, 56 samples from 16 adults and 17 children underwent concurrent Bethesda assay and ELISA screen between November 2010 and June 2013. Since ELISA implementation, 389 ELISA screens and 187 Bethesda assays were performed. ELISA screens resulted in the avoidance of Bethesda assays in 85% (330/389) of samples submitted during that period. Specificities and positive predictive values were lower in the clinical sample due to a larger number of false positives (n=18; 32%) relative to the validation study (n=3; 9%), while sensitivity and negative predictive values remained at 100% (Table 1). Interestingly, 67% (2/3) of false positive ELISA screens in the laboratory setting had Bethesda positive histories; however, in the clinical sample, only 6% (1/18) had a distant history of inhibitors. ODs were available for all validation study samples and for 54 clinical samples. Redefinition of our OD cutoff to improve specificity was prevented by false positive patients with strongly positive ODs. Conclusions The ELISA screen used in this setting is highly sensitive for anti-factor VIII antibody detection in congenital hemophilia A. However, compared to pre-clinical data, our 2.5 years of clinical experience reveals a high incidence of false positives resulting in a significantly lower specificity. Our approximate cost savings in British Columbia as a result of avoided Bethesda assays due to ELISA screens (n=330) is $90 per test, and may exceed $200 per test in the United States. Additional testing and follow-up on positive ELISA results can be an inconvenience in both time and blood sampling, especially in the pediatric population where inhibitor status is more closely monitored. The cost ramifications of this follow up testing could not be quantified in this study. The potential implications for the detection of non-neutralizing anti-FVIII antibodies in hemophilia A need to be further explored and long-term study and monitoring of these discrepant patients is warranted. Disclosures: No relevant conflicts of interest to declare.

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Chin, V., E. Stone, A. Havryk, M. Plit, R. Mccloy, A. Murphy, A. Field, N. Watkins et J. Powell. « P60.01 Single-Cell Transcriptomics to Assess Response to Immunotherapy in Advanced Lung Cancer ex-vivo : Developing a Functional Predictive Assay ». Journal of Thoracic Oncology 16, n^o 3 (mars 2021) : S540—S541. http://dx.doi.org/10.1016/j.jtho.2021.01.960.

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Monforte, Víctor, Piedad Ussetti, Raquel Castejón, Helena Sintes, Virginia Luz Pérez, Rosalía Laporta, Amparo Sole et al. « Predictive Value of Immune Cell Functional Assay for Non-Cytomegalovirus Infection in Lung Transplant Recipients : A Multicenter Prospective Observational Study ». Archivos de Bronconeumología (English Edition) 57, n^o 11 (novembre 2021) : 690–96. http://dx.doi.org/10.1016/j.arbr.2020.12.012.

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Flores, Victor V., Jennifer A. Gifford, Blake K. Wilson et Craig A. Gifford. « PSV-A-4 Novel Method to Identify Cattle Predisposed to Severe Cases of Bovine Respiratory Disease ». Journal of Animal Science 100, Supplement_3 (21 septembre 2022) : 256–57. http://dx.doi.org/10.1093/jas/skac247.464.

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Abstract Respiratory disease in several species can range from subclinical infection to acute death. Bovine respiratory disease (BRD) remains the greatest economic challenge facing the beef industry. Histones are core proteins in chromosomal structure that, when released extracellularly, are cytotoxic and could contribute to exacerbated tissue necrosis during infection. Work in our laboratory demonstrated cattle suffering severe cases of BRD had limited capacity to protect against the cytotoxic effects of extracellular histones, however, the protective mechanism(s) remain unknown. Literature suggests components of the complement system interact with extracellular histones, but the effects on cytotoxic activity of histones is unclear. The objective of the current experiment was to evaluate protective capacity against extracellular histones and complement activity in feedlot heifers and subsequent disease outcomes. Serum samples were collected from 118 heifer calves (BW 229 ± 2.4 kg) at feedlot arrival and evaluated for protective capacity against histone toxicity employing an extracellular histone toxicity assay; and complement activity using a 50% hemolytic complement screening assay. Animals were retrospectively assigned to groups consisting of: calves not requiring treatment with antibiotics for BRD (NT; n = 80) or calves that died from BRD within 1 wk of entering the feedlot (DA; n = 9). Serum from DA animals was less (P < 0.001) protective than NT animals against histone toxicity. Complement activity of DA animals was reduced (P < 0.05) compared to NT animals. Using a ratio of both assays under stringent selection points, the positive predictive value was 75%, and negative predictive value was 94%. Results indicate that cattle predisposed to severe cases of respiratory have impaired complement activity presumably contributing to impaired protective capacity against histone toxicity. Analyzing these functional pathways may provide a novel selection tool against cattle predisposed to severe cases of BRD.

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Salpeter, Seth, Vered Bar, Guy Neev, Adi Zundelevich, Gil Rosen, Sandra Hanks, Naoise Costelloe, Jonathan Krell et Ravid Straussman. « Abstract 4347 : A pivotal clinical trial of cResponse, a functional assay for cancer precision medicine ». Cancer Research 83, n^o 7_Supplement (4 avril 2023) : 4347. http://dx.doi.org/10.1158/1538-7445.am2023-4347.

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Abstract Precision cancer therapy has the potential to revolutionize treatment outcome. While genomic analysis has become central to cancer personalized medicine, recent studies have not shown that it drastically improves the patient’s survival as compared to standard drug selection. Additionally, genomic mutations may suggest several treatment protocols without elucidating which approach will yield the best clinical response. To advance cancer precision guidance, we have developed cResponse, a combined genomic-functional drug sensitivity platform to determine individualized patient treatment regimens. Fresh patient cancer samples are taken by biopsy or resection and sectioned into 250uM slices which when cultured in cResponse platform demonstrate similar architecture and tissue proliferation to those found in vivo. An initial clinical study showed that cResponse can preserve human cancer tissue in 3D culture together with its microenvironment, including endothelial and immune cells, at a high viability (>90%) with continued cell division for more than 7 days. On a cohort of 34 patients treated with neoadjuvant therapy or systemic therapy for metastatic disease, the assay was able to predict patient response to drug treatment with a sensitivity of 96% and a specificity of 77.7%. To further validate the capacity of the cResponse platform to predict patient response to cancer therapy, a follow up pivotal clinical study was established at 7 major cancer centers located in the UK with the goal of recruiting a total of 170 patients to provide a large statistical validation of the previous results. Here we report on the outcome of the first 50 patients recruited to the pivotal trial and describe the predictive results correlated to patient response. Citation Format: Seth Salpeter, Vered Bar, Guy Neev, Adi Zundelevich, Gil Rosen, Sandra Hanks, Naoise Costelloe, Jonathan Krell, Ravid Straussman. A pivotal clinical trial of cResponse, a functional assay for cancer precision medicine. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4347.

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Takaya, Hiroaki, Tadashi Namisaki, Shinya Sato, Kosuke Kaji, Yuki Tsuji, Daisuke Kaya, Yukihisa Fujinaga et al. « Increased Endotoxin Activity Is Associated with the Risk of Developing Acute-on-Chronic Liver Failure ». Journal of Clinical Medicine 9, n^o 5 (14 mai 2020) : 1467. http://dx.doi.org/10.3390/jcm9051467.

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Acute-on-chronic liver failure (ACLF) leads to systematic inflammatory response syndrome and multiple organ failure. This study investigated the relationship between endotoxin (Et) and ACLF with the aim of determining whether Et activity (EA) is useful as a predictive biomarker of ACLF development and whether rifaximin treatment decreased the risk of ACLF development. Two hundred forty-nine patients with liver cirrhosis were enrolled in this study. Et concentration was determined in the whole blood by a semiquantitative EA assay. Predictive factors of ACLF development and the risk of ACLF development with and without rifaximin treatment were identified by univariate and multivariate analysis using Fine and Gray’s proportional subhazards model. EA level was higher in Child-Pugh class B than in class A patients, and class B patients had an increased risk of ACLF development compared with class A patients. Multivariate analysis showed that EA level was a predictive factor independently associated with ACLF development. Rifaximin decreased EA level and the risk of ACLF development in Child-Pugh class B patients. Et levels were associated with functional liver capacity and were predictive of ACLF development in cirrhotic patients. Rifaximin decreased Et level and the risk of ACLF development in advanced cirrhotic patients.

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Willey, Christopher D., Yedeh P. Ying, Anthony B. Morlandt, Hope M. Amm, Patricia H. Hicks, Joshua C. Anderson, Andee M. Beierle et al. « Abstract 4554 : Head and neck cancer HuBiogel-embedded microtumor assay system for therapeutic efficacy testing of patient tumor specimens ». Cancer Research 83, n^o 7_Supplement (4 avril 2023) : 4554. http://dx.doi.org/10.1158/1538-7445.am2023-4554.

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Abstract Head and neck (HN) cancer recurrence is common, and selecting effective salvage systemic therapy remains difficult, particularly for oral cavity cancers. Developing a rapid, robust and predictive therapeutic testing system could support clinical decision-making and improve patient outcomes. We developed a Patient Therapy Evaluation System (PTES) that employs a three-dimensional (3D) fully human microtumor drug testing assay using tissue specimens collected during surgery of HN cancers. Remnant fresh tumor tissue from patients is dissociated into single-cell suspension that is embedded using a novel HuBiogel-cell encapsulation technology (3D microtumors). This high-throughput assay platform allows morphologic, functional, and molecular evaluations in parallel by real-time imaging, cell proliferation, and biomarker protocols. Microtumor viability, growth profiles, and drug screening data are captured at multiple time points up to 14 days. Our initial cohort included 57 patient specimens (53 squamous cell carcinomas, 1 verrucous carcinoma, 1 osteosarcoma, 1 ameloblastoma, and 1 non-cancerous lichenoid mucositis). HuBiogel-embedded tumor cells formed numerous multicellular colonies exhibiting distinct organization and growth patterns in 14-day microtumor cultures. Interestingly, epithelial, stromal and stem-cell like populations were preserved in HN microtumor models based on marker expression. Treatment with single (cisplatin, 5FU, docetaxel) drugs and their combinations resulted in tumor inhibitory responses (IC50) evaluated by CellTiter-Glo assay, and residual surviving cells were also recorded by Calcein-AM staining of 3D Microtumors. While patient-derived HN microtumors were produced with high success rates, factors associated with lower microtumor yield included smaller tumor specimens and low viability after dissociation. In conclusion, our new all human microtumor assay models replicating phenotypic, functional, and molecular properties ex vivo provide a potential theranostic tool for rapidly predicting drug sensitivity and improving treatment strategy for HN cancer patients. Citation Format: Christopher D. Willey, Yedeh P. Ying, Anthony B. Morlandt, Hope M. Amm, Patricia H. Hicks, Joshua C. Anderson, Andee M. Beierle, Carissa M. Thomas, Jason M. Warram, Jingsong Chen, Jeffrey A. Thomas, Katie Banko, Raj K. Singh. Head and neck cancer HuBiogel-embedded microtumor assay system for therapeutic efficacy testing of patient tumor specimens. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4554.

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Churchman, Sarah M., Sally A. Boxall, Dennis McGonagle et Elena A. Jones. « Predicting the Remaining Lifespan and Cultivation-Related Loss of Osteogenic Capacity of Bone Marrow Multipotential Stromal Cells Applicable across a Broad Donor Age Range ». Stem Cells International 2017 (2017) : 1–10. http://dx.doi.org/10.1155/2017/6129596.

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Background and Objectives. Culture expanded multipotential stromal cells (MSCs) have considerable potential for bone regeneration therapy but their wider use is constrained by the lack of simple and predictive assays of functional potency. Extended passaging leads to loss of multipotency but speed of decline depends on MSC donor age. The aim of this study was to develop an assay predictive of MSC culture longevity applicable to a broad donor age range. Materials and Methods. Bone marrow (BM, n=7) was obtained from a diverse range (2–72 years) of healthy donors. MSCs were culture expanded to senescence and their osteoprogenitor content, gene expression profiles, epigenetic signature, and telomere behaviour were measured throughout. Output data was combined for modelling purposes. Results. Regardless of donor age, cultures’ osteoprogenitor content correlated better with remaining lifespan (population doublings before senescence, PD-BS) than proliferative history (accrued PDs). Individual gene’s expression or telomere length did not predict PD-BS but methylation of individual CpG islands did, PRAMEF2 in particular (r=0.775). Coupling the steep relationship of relative SPARC expression with PD-BS (r=-0.753) the formula SPARC × 1/PREMEF2 gave an improved correlation (r=-0.893). Conclusion. A formula based on SPARC mRNA and PRAMEF2 methylation may be used to predict remaining BM-MSC longevity and related loss of multipotentiality independent of donor age.

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Bell, Robin J., Rakibul M. Islam, Marina A. Skiba, Dilinie Herbert, Alejandra Martinez Garcia et Susan R. Davis. « Substituting serum anti-Müllerian hormone for polycystic ovary morphology increases the number of women diagnosed with polycystic ovary syndrome : a community-based cross-sectional study ». Human Reproduction 37, n^o 1 (6 novembre 2021) : 109–18. http://dx.doi.org/10.1093/humrep/deab232.

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Abstract STUDY QUESTION Can serum anti-Müllerian hormone (AMH) replace polycystic ovary morphology (PCOM) determined by ultrasound as a diagnostic component of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER Despite good correlations between serum AMH and PCOM, the use of a high serum AMH as a proxy for PCOM resulted in the reclassification of PCOS in 5% of study participants, with the main effect being more women identified, although some women previously classified as having PCOS were no longer classified as such. WHAT IS KNOWN ALREADY AMH has been proposed as an alternative to PCOM as a diagnostic component of PCOS. Previous studies are limited by poorly defining PCOS, use of infertile women as comparators, measurement of hormones by immunoassay that lack precision in the female range, low-resolution ovarian ultrasound and inconsistent handling and storage of serum samples. STUDY DESIGN, SIZE, DURATION This is an Australian cross-sectional study of 163 non-healthcare-seeking women. PARTICIPANTS/MATERIALS, SETTING, METHODS Serum AMH was measured by both the Ansh picoAMH assay and the Beckman Coulter Access 2 (BA2) assay, in parallel with androgens measured by liquid chromatography–tandem mass spectrometry, in blood samples of women, not pregnant, breast feeding or using systemic steroids, who also underwent high-resolution ovarian ultrasound. PCOS was determined by the Rotterdam criteria with PCOM defined by the Androgen Excess-PCOS Taskforce recommendation of ≥25 follicles in at least one ovary. Cut-off serum concentrations that best identified women as having PCOM were identified by receiver operator characteristic (ROC) curves. MAIN RESULTS AND THE ROLE OF CHANCE A total of 163 women, mean (SD) age 32.5 (5.5) years, who provided a blood sample and had both ovaries visualized on ultrasound were included in the analysis. Women with isolated PCOM had higher median (range) Ansh AMH and BA2 AMH concentrations than those with no PCOS characteristics [56.9 pmol/l (34.6, 104.2) versus 18.7 (3.2, 50.9), P = 0.002 and 38.5 pmol/l (22.2, 100.2) versus 16.7 (3.5, 38.9), P = 0.002, respectively]. An AMH ≥ 44.0 pmol/l, suggested by the ROC curve, identified 80.6% of women with PCOM, falsely identified 15.2% of women without PCOM as having PCOS and had a positive predictive value of 55.6%. The negative predictive value was 94.9%. An AMH BA2 assay cut-off of ≥33.2 pmol/l provided a sensitivity of 80.6%, a specificity of 79.5% and a positive predictive value for PCOM of 48.1%. The negative predictive value was 94.6% for PCOM. When serum AMH was used in the place of PCOM as a diagnostic criterion for PCOS, the Ansh assay resulted in an additional seven women classified as having PCOS and no longer classified one woman as having PCOS. For the BA2 assay, eight additional and two fewer women were classified as having PCOS. Overall, both assays resulted in six more women being classified as having PCOS. LIMITATIONS, REASONS FOR CAUTION Women with functional hypogonadotrophic hypogonadism were not excluded and may have been misclassified as having an oligo-amenorrhoea-PCOM phenotype. As study participants were predominantly Caucasian/White, our findings cannot be generalized to women of other ethnicities. WIDER IMPLICATIONS OF THE FINDINGS Although serum AMH reflects the number of developing ovarian follicles, the absolute values vary between assays and specific reference ranges for individual assays are required. Irrespective of the assay used, replacing PCOM with serum AMH to diagnose PCOS in a community-based sample altered the number of women classified as having or not having PCOS. Consequently, although overall the risk of women being identified as having PCOS would be increased, some women would no longer be classified as having this condition. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by the Norman Beischer Research Foundation and the Grollo-Ruzzene Foundation. S.R.D. is an NHMRC Senior Principal Research Fellow (Grant No. 1135843). S.R.D. reports unrelated support that includes grants from the NHMRC Australia, personal fees for educational activities from Besins Healthcare, Abbott Chile, BioFemme and Pfizer Australia, personal Advisory Board/consultancy fees from Theramex, Abbott Laboratories, Astellas, Mayne Pharmaceuticals, Roche Diagnostics, Lawley Pharmaceuticals and Que Oncology and has received institutional grant funding from Que Oncology and Ovoca research. The other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.

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Margossian, Astrid, Annie Richardson, Michael Churchill, Franz Schaub, Rachele Rosati, Lauren Appleyard, Madison Pollastro et al. « Predictive value of a CLIA-approved organoid based drug sensitivity test. » Journal of Clinical Oncology 38, n^o 15_suppl (20 mai 2020) : 3630. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.3630.

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3630 Background: Precision medicine integrates genetic, molecular, and clinical information to optimize therapy selection for cancer patients. Ex vivo drug testing has the potential to match the right drug to the right patient. We developed a CLIA-certified functional drug assay for all solid tumors which provides an actionable report of organoid sensitivity to targeted, endocrine and chemotherapy agents as a tool for therapeutic decisions. Objectives: To establish the predictive power of the test in relation to well-known genomic biomarkers as well as prior treatments to identify drug sensitivity. To demonstrate that functional drug testing increases the actionability of genomic reports. Methods: From 2016 to 2019, 240 organoids from cancer patients were subjected to functional testing at SEngine Precision Medicine. Patients with advanced primary or metastatic cancer (solid tumors) who were treatment naïve or had previous therapies fail. Fresh samples of tumor cells from core biopsies, surgical excisions, or fluids arrived <48 hrs following collection and were cultured as 3D organoids. They were evaluated using a multi-dose response format with a library of up to 130 compounds. Drug sensitivity was quantified using a score that combines sensitivity and personalization of each patient’s response relative to a reference population. Known genomic actionability from levels of evidence 1-2 from MSKCC OncoKB were queried against results for correlation. Results: Organoids were derived from breast (18.7%), ovarian (18.3%), colorectal (17.9%), pancreatic (6.7%), and others solid tumors (38,3%). Median age of patients was 53 (r5-83). 68 drugs on average were tested per patient with a mean turnaround time of 18 days (r9 -37). A mean of 7 drugs per patient were identified as top scoring drugs. In 75 patients with genomic data, we found high concordance of drug sensitivity with known genomic anchors (e.g., inhibitors of BRCA1/ PARP, ERBB2/HER2, FGFR1-2/FGFR, KRAS, PIK3CA/PI3K), measured as sensitivity to drugs among this targeted groups. However, several patient samples demonstrated sensitivity to targeted agents in the absence of known genomic biomarkers. Most important, analysis of previous treatments indicated >90% of retrospective concordance. Conclusions: Organoid based drug testing exhibits strong concordance with genomic and retrospective clinical evidence. In addition, functional testing identifies candidate therapies in patients with no known biomarkers and can identify the significance of variants currently not validated.

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