Microfluidic active loading of single cells enables analysis of complex clinical specimens

A fundamental trade-off between flow rate and measurement precision limits performance of many single-cell detection strategies, especially for applications that require biophysical measurements from living cells within complex and low-input samples. To address this, we introduce ‘active loading’, an automated, optically-triggered fluidic system that improves measurement throughput and robustness by controlling entry of individual cells into a measurement channel. We apply active loading to samples over a range of concentrations (1–1000 particles μL[superscript −1]), demonstrate that measurement time can be decreased by up to 20-fold, and show theoretically that performance of some types of existing single-cell microfluidic devices can be improved by implementing active loading. Finally, we demonstrate how active loading improves clinical feasibility for acute, single-cell drug sensitivity measurements by deploying it to a preclinical setting where we assess patient samples from normal brain, primary and metastatic brain cancers containing a complex, difficult-to-measure mixture of confounding biological debris.National Cancer Institute (U.S.) (R01 CA170592)National Cancer Institute (U.S.) (R33 CA191143)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)Bridge Projec

Microfluidic active loading of single cells enables analysis of complex clinical specimens

A fundamental trade-off between flow rate and measurement precision limits performance of many single-cell detection strategies, especially for applications that require biophysical measurements from living cells within complex and low-input samples. To address this, we introduce ‘active loading’, an automated, optically-triggered fluidic system that improves measurement throughput and robustness by controlling entry of individual cells into a measurement channel. We apply active loading to samples over a range of concentrations (1–1000 particles μL[superscript −1]), demonstrate that measurement time can be decreased by up to 20-fold, and show theoretically that performance of some types of existing single-cell microfluidic devices can be improved by implementing active loading. Finally, we demonstrate how active loading improves clinical feasibility for acute, single-cell drug sensitivity measurements by deploying it to a preclinical setting where we assess patient samples from normal brain, primary and metastatic brain cancers containing a complex, difficult-to-measure mixture of confounding biological debris.National Cancer Institute (U.S.) (R01 CA170592)National Cancer Institute (U.S.) (R33 CA191143)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)Bridge Projec

EANO guideline on rational molecular testing of gliomas, glioneuronal, and neuronal tumors in adults for targeted therapy selection

The mainstay of treatment for adult patients with gliomas, glioneuronal and neuronal tumors consists of combinations of surgery, radiotherapy, and chemotherapy. For many systemic cancers, targeted treatments are a part of the standard of care, however, the predictive significance of most of these targets in central nervous system (CNS) tumors remains less well-studied. Despite that, there is increasing use of advanced molecular diagnostics that identify potential targets, and tumor-agnostic regulatory approvals on targets also present in CNS tumors have been granted. This raises the question of when and for which targets it is meaningful to test in adult patients with CNS tumors. This evidence-based guideline reviews the evidence available for targeted treatment for alterations in the RAS/MAPK pathway (BRAF, NF1), in growth factor receptors (EGFR, ALK, fibroblast growth factor receptor (FGFR), neurotrophic tyrosine receptor kinase (NTRK), platelet-derived growth factor receptor alpha, and ROS1), in cell cycle signaling (CDK4/6, MDM2/4, and TSC1/2) and altered genomic stability (mismatch repair, POLE, high tumor mutational burden (TMB), homologous recombination deficiency) in adult patients with gliomas, glioneuronal and neuronal tumors. At present, targeted treatment for BRAF p.V600E alterations is to be considered part of the standard of care for patients with recurrent gliomas, pending regulatory approval. For approved tumor agnostic treatments for NTRK fusions and high TMB, the evidence for efficacy in adult patients with CNS tumors is very limited, and treatment should preferably be given within prospective clinical registries and trials. For targeted treatment of CNS tumors with FGFR fusions or mutations, clinical trials are ongoing to confirm modest activity so far observed in basket trials. For all other reviewed targets, evidence of benefit in CNS tumors is currently lacking, and testing/treatment should be in the context of available clinical trials

The biological significance of tumor grade, age, enhancement, and extent of resection in IDH-mutant gliomas:How should they inform treatment decisions in the era of IDH inhibitors?

The 2016 and 2021 World Health Organization 2021 Classification of central nervous system tumors have resulted in a major improvement in the classification of isocitrate dehydrogenase (IDH)-mutant gliomas. With more effective treatments many patients experience prolonged survival. However, treatment guidelines are often still based on information from historical series comprising both patients with IDH wild-type and IDH-mutant tumors. They provide recommendations for radiotherapy and chemotherapy for so-called high-risk patients, usually based on residual tumor after surgery and age over 40. More up-to-date studies give a better insight into clinical, radiological, and molecular factors associated with the outcome of patients with IDH-mutant glioma. These insights should be used today for risk stratification and for treatment decisions. In many patients with IDH-mutant grades 2 and 3 glioma, if carefully monitored postponing radiotherapy and chemotherapy is safe, and will not jeopardize the overall outcome of patients. With the INDIGO trial showing patient benefit from the IDH inhibitor vorasidenib, there is a sizable population in which it seems reasonable to try this class of agents before recommending radio-chemotherapy with its delayed adverse event profile affecting quality of survival. Ongoing trials should help to further identify the patients that are benefiting from this treatment.</p

Epstein-Barr Virus and immune status imprint the immunogenomics of non-Hodgkin lymphomas occurring in immune-suppressed environments

Non-Hodgkin lymphomas (NHL) commonly occur in immune-deficient (ID) patients, both HIV-infected and transplanted, and are often EBV-driven with cerebral localization, raising the question of tumor immunogenicity, a critical issue for treatment responses. We investigated the immunogenomics of 68 lymphoproliferative disorders from 51 ID (34 posttransplant, 17 HIV+) and 17 immunocompetent patients. Overall, 72% were Large B Cells Lymphoma (LBCL) and 25% were primary central-nervous-system lymphoma (PCNSL) while 40% were EBV-positive. Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline allowed analysis of tumor mutational burden (TMB), tumor landscape and microenvironment (TME) and prediction of tumor neoepitopes. Both TMB (2.2 vs 3.4/Mb, p=0.001) and neoepitopes numbers (40 vs 200, p=0.00019) were lower in EBVpositive than in EBV-negative NHL, regardless of the immune status. In contrast both EBV and the immune status influenced the tumor mutational profile, with HNRNPF and STAT3 mutations exclusively observed in EBV-positive and ID NHL, respectively. Peripheral blood T-cell responses against tumor neoepitopes were detected in all EBV-negative cases but in only half EBV-positive ones, including responses against IgH-derived MHC-class-II restricted neoepitopes. The TME analysis showed higher CD8 T cell infiltrates in EBVpositive vs EBV-negative NHL, together with a more tolerogenic profile composed of Tregs, type-M2 macrophages and an increased expression of negative immune-regulators. Our results highlight that the immunogenomics of NHL in patients with immunodeficiency primarily relies on the tumor EBV status, while T cell recognition of tumor- and IgH-specific neoepitopes is conserved in EBV-negative patients, offering potential opportunities for future T cell-based immune therapies

Cost of antimicrobial resistance in France : assessment from the medical-administrative databases

La résistance aux antibiotiques est considérée comme une menace majeure pour la santé publique mondiale. Le traitement des infections à germe résistant est plus difficile et conduit à des modifications de l’organisation des soins avec une augmentation des durées de séjour, de la morbidité et de la mortalité. L’évaluation du poids économique de la résistance aux antibiotiques pourrait éclairer les décideurs sur les priorités d’action à mener en termes de prévention, de recherche et de prise en charge. L’objectif général de cette thèse est d’évaluer l’impact économique de la résistance aux antibiotiques à partir des données du Système National des Données de Santé (SNDS). L’utilisation du SNDS permet d’identifier de façon exhaustive les patients hospitalisés atteints d’une infection bactérienne, suivis ou non par les professionnels libéraux ou en structure adaptée. Le suivi des séjours hospitaliers avec les consultations en médecine de ville rend possible la reconstitution des parcours de soins et les coûts qui y sont associés. La population étudiée concerne l’ensemble des patients hospitalisés présentant une infection bactérienne aiguë (regroupé en 13 sites infectieux). Dans un premier travail, nous nous sommes intéressés au coût hospitalier, du point de vue du payeur. Une étude cas-témoins appariés a permis d’estimer un surcoût hospitalier attribuable à la résistance de 110 millions € en 2015 et une extrapolation conduit à un surcoût de 290 millions €. Dans un second temps, nous nous sommes concentrés sur les conséquences à 12 mois d’une hospitalisation à germe résistant aux travers de quatre études : (1) Pour les patients ayant eu une infection à germe résistant, une analyse de séquence a permis d’identifier cinq parcours hospitaliers types. Les parcours hospitaliers les plus longs étaient observés suite à une infection ostéo-articulaire et une mortalité élevée concernait principalement les infections du cœur et du médiastin ou des voies respiratoires basses ; (2) La dépense ambulatoire étudiée par une approche de double différence, a montré que la surconsommation attribuable à la résistance aux antibiotiques était faible et limitée au premier mois qui suit une hospitalisation ; (3) La consommation de ressources hospitalières mesurée par la durée d’hospitalisations (en jours) attribuable à l’antibiorésistance est augmentée pour deux secteurs : en séjours hospitaliers en soins de courte durée pour infection et en hospitalisation à domicile ; (4) le surcoût hospitalier attribuable à la résistance aux antibiotiques l’année qui suit l’hospitalisation initiale a été estimé à 618 € [IC95% 419 ; 817] par patient. À travers 5 indicateurs économiques, cette thèse a mise en évidence que l’antibiorésistance provoque un coût substantiel pour l’assurance maladie.Antimicrobial resistance (AMR) is a major threat to global public health, makes infections more difficult to treat, and potentially jeopardizes medical progress and innovation. AMR is also associated with higher morbidity and mortality. Assessing the economic burden of AMR could highlight priorities in prevention, research and management for decision-makers. The main objective of this Ph.D. dissertation is to assess the economic impact of AMR in France based on data from the National Health Data System (SNDS) database. SNDS contains patient-level medical data and inpatient and outpatient care costs reimbursed by national health insurance. We thus used SNDS to analyse care pathways and associated costs among a population that included all hospitalized patients with acute bacterial infection (classified into 13 infectious sites). First, we investigated the hospital cost from the payer's perspective. Through a matched case-control design, we estimated an additional hospital cost of €110 million caused by AMR in 2015, with an extrapolation showing that the overall cost could reach €290 million. Second, we focused on the effects at 12 months of hospitalization with AMR. In this context, four studies were developed. (1) For patients with resistant infections, a sequence analysis identified five distinct hospital pathways. Longest hospital stays were observed for bone and joint infections, whereas patients with heart and mediastinum infections or lower respiratory tract infections had higher mortality rates. (2) Ambulatory expenditure was studied using a difference-in-difference approach and we showed a low overconsumption due to AMR, limited to the first month following hospitalization. (3) Hospital resource consumption measured by duration of hospitalization due to AMR was increased in acute care hospital stays for infection and in hospitalization at home. (4) Additional hospital cost due to antibiotic resistance during the year following initial hospitalization was estimated at €618 [IC95% 419; 817] per patient. In conclusion, using five economic criteria this Ph.D dissertation has shown that AMR bears a substantial cost burden on the French public health insurance system

Coût de l’antibiorésistance en France : évaluation à partir des bases de données médico-administratives

Antimicrobial resistance (AMR) is a major threat to global public health, makes infections more difficult to treat, and potentially jeopardizes medical progress and innovation. AMR is also associated with higher morbidity and mortality. Assessing the economic burden of AMR could highlight priorities in prevention, research and management for decision-makers. The main objective of this Ph.D. dissertation is to assess the economic impact of AMR in France based on data from the National Health Data System (SNDS) database. SNDS contains patient-level medical data and inpatient and outpatient care costs reimbursed by national health insurance. We thus used SNDS to analyse care pathways and associated costs among a population that included all hospitalized patients with acute bacterial infection (classified into 13 infectious sites). First, we investigated the hospital cost from the payer's perspective. Through a matched case-control design, we estimated an additional hospital cost of €110 million caused by AMR in 2015, with an extrapolation showing that the overall cost could reach €290 million. Second, we focused on the effects at 12 months of hospitalization with AMR. In this context, four studies were developed. (1) For patients with resistant infections, a sequence analysis identified five distinct hospital pathways. Longest hospital stays were observed for bone and joint infections, whereas patients with heart and mediastinum infections or lower respiratory tract infections had higher mortality rates. (2) Ambulatory expenditure was studied using a difference-in-difference approach and we showed a low overconsumption due to AMR, limited to the first month following hospitalization. (3) Hospital resource consumption measured by duration of hospitalization due to AMR was increased in acute care hospital stays for infection and in hospitalization at home. (4) Additional hospital cost due to antibiotic resistance during the year following initial hospitalization was estimated at €618 [IC95% 419; 817] per patient. In conclusion, using five economic criteria this Ph.D dissertation has shown that AMR bears a substantial cost burden on the French public health insurance system.La résistance aux antibiotiques est considérée comme une menace majeure pour la santé publique mondiale. Le traitement des infections à germe résistant est plus difficile et conduit à des modifications de l’organisation des soins avec une augmentation des durées de séjour, de la morbidité et de la mortalité. L’évaluation du poids économique de la résistance aux antibiotiques pourrait éclairer les décideurs sur les priorités d’action à mener en termes de prévention, de recherche et de prise en charge. L’objectif général de cette thèse est d’évaluer l’impact économique de la résistance aux antibiotiques à partir des données du Système National des Données de Santé (SNDS). L’utilisation du SNDS permet d’identifier de façon exhaustive les patients hospitalisés atteints d’une infection bactérienne, suivis ou non par les professionnels libéraux ou en structure adaptée. Le suivi des séjours hospitaliers avec les consultations en médecine de ville rend possible la reconstitution des parcours de soins et les coûts qui y sont associés. La population étudiée concerne l’ensemble des patients hospitalisés présentant une infection bactérienne aiguë (regroupé en 13 sites infectieux). Dans un premier travail, nous nous sommes intéressés au coût hospitalier, du point de vue du payeur. Une étude cas-témoins appariés a permis d’estimer un surcoût hospitalier attribuable à la résistance de 110 millions € en 2015 et une extrapolation conduit à un surcoût de 290 millions €. Dans un second temps, nous nous sommes concentrés sur les conséquences à 12 mois d’une hospitalisation à germe résistant aux travers de quatre études : (1) Pour les patients ayant eu une infection à germe résistant, une analyse de séquence a permis d’identifier cinq parcours hospitaliers types. Les parcours hospitaliers les plus longs étaient observés suite à une infection ostéo-articulaire et une mortalité élevée concernait principalement les infections du cœur et du médiastin ou des voies respiratoires basses ; (2) La dépense ambulatoire étudiée par une approche de double différence, a montré que la surconsommation attribuable à la résistance aux antibiotiques était faible et limitée au premier mois qui suit une hospitalisation ; (3) La consommation de ressources hospitalières mesurée par la durée d’hospitalisations (en jours) attribuable à l’antibiorésistance est augmentée pour deux secteurs : en séjours hospitaliers en soins de courte durée pour infection et en hospitalisation à domicile ; (4) le surcoût hospitalier attribuable à la résistance aux antibiotiques l’année qui suit l’hospitalisation initiale a été estimé à 618 € [IC95% 419 ; 817] par patient. À travers 5 indicateurs économiques, cette thèse a mise en évidence que l’antibiorésistance provoque un coût substantiel pour l’assurance maladie

Mutational burden and immune recognition of gliomas

International audiencePurpose of review Recent evidence suggests high tumor mutational burden (TMB-H) as a predictor of response to immune checkpoint blockade (ICB) in cancer. However, results in TMB-H gliomas have been inconsistent. In this article, we discuss the main pathways leading to TMB-H in glioma and how these might affect immunotherapy response. Recent findings Recent characterization of TMB-H gliomas showed that "post-treatment hypermutation" related to mismatch repair (MMR) deficiency is the most common mechanism leading to TMB-H in gliomas. Unexpectedly, preliminary evidence suggested no benefit with ICB as compared to chemotherapy in this population. In contrast, ICB response was reported in a subset of TMB-H gliomas associated with constitutional MMR or polymerase epsilon (POLE) defects (e.g., constitutional biallelic MMRd deficiency). In other cancers, several trials suggest increased ICB efficacy is critically associated with increased lymphocyte infiltration at baseline which is missing in most gliomas. Further characterization of the immune microenvironment of gliomas is needed to identify biomarkers to select the patients who will benefit from ICB