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

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

Development of different analytical techniques to investigate radionuclides difficult to measure and to improve detection limits.

International audienceThe radioactive waste management is a challenging task faced by nuclear powercountries and is a prime concern for the public and therefore for control authorities. In France,several nuclear facilities were shut down few years ago and are now under dismantling. In thesefacilities, after the disposal of different equipments (glove boxes etc.) and a cleaning-up step,laboratories are subject to decommissioning and sometimes demolition. In order to establish theworking procedure of dismantling, an inventory on the radioactive level of the various materialsand areas is essential. Furthermore, correct waste classification is crucial to define the appropriatefinal disposal repository and also to keep disposal cost down to a minimum. For all thesecharacterizations, the laboratory in CEA Saclay (LASE) develops radiochemical procedures andanalytical techniques to provide results with high confidence level and with very low detectionlimits

Developpement de nouvelles methodes en vue de la quantification de radionucleides difficilement mesurables dans des dechets nucleaires et amelioration des limites de detection

National audienceAu sein du CEA Saclay, le Laboratoire d'Analyse en Soutien aux Exploitants (LASE) situe au batiment 459 est specialise dans l'analyse de Radionucleides difficilement mesurables dans les dechets nucleaires de faible et moyenne activite. Une vingtaine de modes operatoires existent au laboratoire pour analyser les differents Radionucleides, tels que le 3H, le 14C, le 36Cl, le 63Ni, le 108mAg ou l'129I. En constante evolution, ces protocoles peuvent etre appliques a de nombreux types de materiaux (solides, liquides, boues) et l'utilisation de traceurs permet de suivre les mises en solution et les multiples etapes des separations radiochimiques indispensables pour la mesure de ces emetteurs. Apres une breve presentation generale de la methodologie d'analyse des Radionucleides difficilement mesurables, quelques exemples seront presentes.En tant qu'emetteurs beta, le 3H et le 14C sont mesures au laboratoire par scintillation liquide apres une etape de combustion permettant d'eliminer les interferents. Le LASE s'est recemment dote de deux fours a pyrolyse multi-tubes afin d'accroitre sa capacite d'analyses du 3H et du 14C face a l'accroissement des demandes liees aux demantelements des installations nucleaires. Ce nouveau protocole est en cours de validation a partir de la preparation de betons dopes en 3H et 14C et d'une comparaison avec un laboratoire partenaire.En tant qu'emetteur beta, le 36Cl est analyse au laboratoire par scintillation liquide apres un protocole radiochimique base sur la synthese organique d'une molecule marquee au 36Cl. En raison de la limite maximale d'acceptabilite peu elevee (5 Bq/g) au centre de l'Aube pour le 36Cl, le LASE s'est oriente vers la spectrometrie de masse par accelerateur (SMA) comme technique alternative de detection. La SMA a ete recemment appliquee avec succes a l'analyse d'echantillons d'aciers actives permettant de quantifier des teneurs en 36Cl de l'ordre de 10-3 Bq/g. L'129I contenu dans des bitumes a aussi ete analyse par SMA.En raison de l'evolution des reglementations dans le domaine des risques chimiques et notamment de la directive REACH, des composes, tels que le chloroforme, doivent etre progressivement elimines des modes operatoires. La methode de reference du 63Ni est basee sur une extraction liquide-liquide avec du chloroforme. Le LASE a ainsi developpe un protocole alternatif reposant sur une purification du 63Ni par resine d'extraction chromatographique dite resine Ni. Ce mode operatoire permet d'atteindre une selectivite et une efficacite equivalentes a celles du mode operatoire de reference.Recemment, le LASE a egalement mis au point une methode d'analyse du 126Sn dans les dechets nucleaires. Apres purification, le 126Sn peut etre detecte par ICP-MS Q a des teneurs de l'ordre de 10-1 Bq/g. Afin de verifier l'homogeneite des echantillons et d'eviter leurs mises en solution fastidieuses, le LASE developpe aussi des mesures par spectrometrie gamma sur solides. Cette technique permet d'atteindre une meilleure representativite et de diminuer les limites de detection d'un facteur 1 a 100

Capabilities of LIBS for analysis of geological samples at stand-off distances in a Mars atmosphere

The use of LIBS for stand-off elemental analysis of geological and other samples in a simulated Mars atmosphere is being evaluated. Analytical capabilities, matrix effects, and other factors effecting analysis are being determined. Through funding from NASA's Mars Instrument Development Program (MIDP), we have been evaluating the use of LIBS for future use on landers and rovers to Mars. Of particular interest is the use of LIBS for stand-off measurements of geological samples up to 20 meters from the instrument. Very preliminary work on such remote LIBS measurements based on large laboratory type equipment was carried out about a decade ago. Recent work has characterized the capabilities using more compact instrumentation and some measurements have been conducted with LIBS on a NASA rover testbed

Why nuclear wastes characterization require intercomparaisons ?

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