Tumours with loss of MSH6 expression are MSI-H when screened with a pentaplex of five mononucleotide repeats

Contains fulltext : 87589.pdf (publisher's version ) (Closed access)BACKGROUND: microsatellite instability (MSI) is commonly screened using a panel of two mononucleotide and three dinucleotide repeats as recommended by a consensus meeting on MSI tumours held at the National Cancer Institute (Bethesda, MD, USA). According to these recommendations, tumours are classified as MSI-H when at least two of the five microsatellite markers show instability, MSI-L when only one marker shows instability and MSS when none of the markers show instability. Almost all MSI-H tumours are characterised by alterations in one of the four major proteins of the mismatch repair (MMR) system (MLH1, MSH2, MSH6 or PMS2) that renders them MMR deficient, whereas MSI-L and MSS tumours are generally MMR proficient. However, tumours from patients with a pathogenic germline mutation in MSH6 can sometimes present an MSI-L phenotype with the NCI panel. The MSH6 protein is not involved in the repair of mismatches of two nucleotides in length and consequently the three dinucleotide repeats of the NCI panel often show stability in MSH6-deficient tumours. METHODS: a pentaplex panel comprising five mononucleotide repeats has been recommended as an alternative to the NCI panel to determine tumour MSI status. Several studies have confirmed the sensitivity, specificity and ease of use of the pentaplex panel; however, its sensitivity for the detection of MSH6-deficient tumours is so far unknown. Here, we used the pentaplex panel to evaluate MSI status in 29 tumours known to harbour an MSH6 defect. RESULTS: MSI-H status was confirmed in 15 out of 15 (100%) cases where matching normal DNA was available and in 28 out of 29 (97%) cases where matching DNA was not available or was not analysed. CONCLUSION: these results show that the pentaplex assay efficiently discriminates the MSI status of tumours with an MSH6 defect

Nonsense-Mediated mRNA Decay Impacts MSI-Driven Carcinogenesis and Anti-Tumor Immunity in Colorectal Cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3ε-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2×10−16). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs

Rapid Measurements by Autoradiography to improve sampling process required for Radiochemical Analysis

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Do we really need very low level measurement for long life radionuclide in nuclear waste?

International audienceWaste management is a key issue for nuclear industry. In France, ANDRA (the national agency for nuclear waste management) is in charge of sizing, building and operating of waste repositories. In order to guarantee the security of these facilities, a list of radionuclides and their maximum associated activities has been defined as acceptance criteria by ANDRA. From this list, some are readily and easily measured while many, such as pure beta emitters are difficult to measure, requiring long radiochemical processes. In several cases, result of the measurements carried out, after radiochemical process, with Liquid Scintillation Counting (LSC) detection technique is often under the detection limit of typically 1 Bq/g. In the lack of other data, stakeholder automatically retain the detection limit as an overestimation of waste activity. Yet, to overestimate waste activities can dramatically increase the cost of their management and reduce the storage capacity of the facilities. To overcome this problem, the Operator Support Analyses Laboratory has developed new radioanalytical procedures to measure radionuclides of interest (Cl-36, I-129, Ca-41) by Accelerator Mass Spectrometry (AMS) at level as low as 1 mBq/g in nuclear waste matrices. We present here the challenges encountered to reach such activity levels. Radiochemical blank and homemade standard have been analyzed to calibrate the analytical procedure. Challenges in term of blank experiments and contamination check procedures in order to ensure such activity levels will be discussed

La microfluidique au service de l’analyse des radionucléides

International audienceLa microfluidique se révèle aujourd'hui comme un des domaines scientifiques et techniques les plus dynamiques pour de nombreuses applications liées aux sciences chimiques, biologiques et analytiques. Plus particulièrement, dans le domaine de la chimie, la microfluidique offre des perspectives nouvelles suscitées par des objectifs de sécurisation, d'optimisation et de diversification des procédés (reduction des transports de matière, reduction des quantités stockées, énergétique, conditions réactionnelles inaccessibles aux réacteurs macroscopiques). Cette échelle réduite permet de concevoir des dispositifs qui satisfont la plupart des critères de la chimie verte. Dans le domaine de l'analyse chimique, cela se traduit par une reduction très importante des quantités d’échantillons, des réactifs, des déchets générés par les installations, du temps d'analyse, par l'utilisation de microsystèmes ou « laboratoire-sur-puce ». Pour des échantillons difficiles a manipuler ou hostiles, par exemple radioactifs, les microsystèmes analytiques offrent de nombreux atouts alliant performances et sûreté avec un enjeu de durabilité des procédés industriels qui s'appuient sur des analyses de plus en plus nombreuses.D'une manière générale, l’idée consiste a intégrer dans un dispositif miniaturise les fonctions nécessaires a l'analyse d'un échantillon : déplacement des fluides, gestion des flux, melange, reaction, filtration, separation, pré-concentration, contrôles thermiques et detection des espèces chimiques. Actuellement, les applications industrielles couvrent les domaines du diagnostic génétique et medical, de la pharmacie, de l'agro-alimentaire, de l'environnement mais peu d'etudes concernent le nucléaire et en particulier l'aval du cycle du combustible. Parmi les travaux menés au CEA/DPC depuis plusieurs années, des microsystèmes intégrant un monolithe organique fonctionnalisé sont développés dans le but de réaliser des separations chimiques chromatographiques d'elements cibles, comme l'uranium, pour les analyser dans des milieux acides concentres. Ces microsystèmes sont ensuite opérés sur une plateforme dite « lab-on-a-CD » qui va permettre le déplacement contrôle des fluides sans connectiques. Ces dispositifs analytiques innovants, automatisables, doivent permettre un gain très important pour les laboratoires d'analyse en termes de cadence d'analyse et de facilite de manipulation d’échantillons radioactifs, avec l'objectif de répondre aux exigences de la chimie verte

Anion exchange microsystems for radionuclides separation in nitric acid media

International audienceThe use of a centrifugal microfluidic platform is an alternative to classical chromatographic procedures for radiochemistry. The original design of the microfluidic platform has been thought to fasten and simplify the prototyping process with the use of a circular platform integrating four rectangular microchips made of thermoplastic. The microchips, dedicated to anion-exchange chromatographic separations, integrate a localized monolithic stationary phase as well as injection and collection reservoirs. An ion-exchange support with respect to the in-situ light-addressable process of elaboration is specifically designed to be incorporated as radiochemical sample preparation module in microsystem devices. Since their introduction in the early 1990s polymethacrylate monoliths have emerged as a powerful alternative for microscale separations or sample treatment. Their relatively simple implementation in columns with small internal diameters makes them particularly attractive for the new chromatographic challenges of complex matrices analysis and on-chip separations. Despite their relatively poor ion-exchange capacity due to their highly porous structure, their use as anion exchangers is of large interest for nuclear analysis as numerous separations are based on this process. A photopolymerized poly(ethylene glycol dimethacrylate-co-allyl methacrylate) monolith has been synthesized. It was then functionalized via a photografting process based on the ene-thiol click chemistry to give anion exchange properties to the monolith. The stationary phase has been optimized to improve its chromatographic performances especially regarding anion exchange chromatography. Synthesis and chromatographic results obtained with this monolith at macro scale and in COC micro-devices will be presented and discuss in term of reproducibility and robustness. Application to analysis of nuclear material will be presented.Thanks to their unique easy-to-use features, centrifugal microfluidic platforms are potential successful candidates for the downscaling of chromatographic separation and especially in the case of radioactive samples. Automation, multiplexing, easy integration in glove-boxes environment and low cost of maintenance are some advantages of the development of centrifugal microfluidic devices. Moreover the low amount of sample needed for the analysis allows the decrease of volume of effluents and the exposition of the operators to radiations during the analysis

Microfluidic Systems for Radiochemistry Synthesis and Functionalization of Polymer Monoliths for Chromatographic Separation of U and Eu

International audienceThe use of a centrifugal microfluidic platform is an alternative to classical chromatographic procedures for radiochemistry. The original design of the microfluidic platform has been thought to fasten and simplify the prototyping process with the use of a circular platform integrating four rectangular microchips made of thermoplastic. The microchips, dedicated to anion-exchange chromatographic separations, integrate a localized monolithic stationary phase as well as injection and collection reservoirs. An ion-exchange support with respect to the in-situ light-addressable process of elaboration is specifically designed to be incorporated as radiochemical sample preparation module in microsystem devices. Since their introduction in the early 1990s polymethacrylate monoliths have emerged as a powerful alternative for microscale separations or sample treatment. Their relatively simple implementation in columns with small internal diameters makes them particularly attractive for the new chromatographic challenges of complex matrices analysis and on-chip separations. Despite their relatively poor ion-exchange capacity due to their highly porous structure, their use as anion exchangers is of large interest for nuclear analysis as numerous separations are based on this process. A photopolymerized poly(ethylene glycol dimethacrylate-co-allyl methacrylate) monolith has been synthesized and a photografting process based on the ene-thiol click chemistry has been performed to give anion exchange properties to the monolith. The stationary phase has been optimized to improve its chromatographic performances especially regarding anion exchange chromatography. Synthesis and chromatographic results obtained with this monolith in COC micro-devices will be presented and discuss in term of reproducibility and robustness. Application to analysis of nuclear material will be presented.Thanks to their unique easy-to-use features, centrifugal microfluidic platforms are potential successful candidates for the downscaling of chromatographic separation of radioactive samples (automation, multiplexing, easy integration in glove-boxes environment and low cost of maintenance)

Liquid-liquid extraction of two radiochemical systems at micro-scale predict and achieve segmented flow to optimize mass transfer

International audienceOne of the most important separation techniques in radiochemical procedures is solvent extraction. In the last decade, a growing interest in its use in microsystems with multiphase micro-flows has emerged because such systems allow a good control of the interface area between aqueous and organic phases as well as of the contact time of the two phases.A previous study was devoted to the micro-extraction in parallel flows of uranium in chloride media by Aliquat 336 and europium in nitric acid by the N,N'-dimethyl N,N'-dibutyl tetradecylmalonamide. While in the first case an optimal extraction yield could be obtained, slower reaction kinetics prevented the second chemical system extraction from being complete. A way to improve mass transfer for slow systems is to increase the specific interfacial area. We therefore investigated liquid-liquid extraction for the same chemical systems by implementing segmented flows. Both the internal circulation stimulated within droplets by their passage along micro-channels and the increase in the interfacial area are responsible for a large enhancement in the interfacial mass transfer and reaction rate. Therefore, an improvement in liquid-liquid extraction yield could be expected. Based on previous works from Van Steijn and Xu, a numerical model was developed to accurately predict the characteristics of droplet production at a T-junction (volumes, frequency, spacing, and specific interfacial area) for the two aforementioned chemical systems. Using parameters screening, the respective influence of liquids viscosities, dimensional parameters and flow rates was evidenced. This numerical model was verified experimentally by generating on-chip segmented flows, and offered ways of improvement in both operating parameters and chip design. Finally, phase separation was obtained using selective membrane, and radionuclides micro-extractions were performed. Micro-extraction yields were compared to conventional batch extraction

H-3 measurement in radioactive wastes Efficiency of the pyrolysis method to extract tritium from aqueous effluent, oil and concrete.

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One step U-Pu-Cs-Ln-steel separation using TRU preconditioned extraction resins from Eichrom for application on transmutation targets

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