Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform

Extracellular vesicles (EVs) are double-layered phospholipid membrane vesicles that are released by most cells and can mediate intercellular communication through their RNA cargo. In this study, we tested if the NanoString nCounter platform can be used for the analysis of EV-mRNA. We developed and optimized a methodology for EV enrichment, EV-RNA extraction and nCounter analysis. Then, we demonstrated the validity of our workflow by analyzing EV-RNA profiles from the plasma of 19 cancer patients and 10 controls and developing a gene signature to differentiate cancer versus control samples. TRI reagent outperformed automated RNA extraction and, although lower plasma input is feasible, 500 μL provided highest total counts and number of transcripts detected. A 10-cycle pre-amplification followed by DNase treatment yielded reproducible mRNA target detection. However, appropriate probe design to prevent genomic DNA binding is preferred. A gene signature, created using a bioinformatic algorithm, was able to distinguish between control and cancer EV-mRNA profiles with an area under the ROC curve of 0.99. Hence, the nCounter platform can be used to detect mRNA targets and develop gene signatures from plasma-derived EVs

BRAF mutations classes I, II, and III in NSCLC patients included in the SLLIP trial : The need for a new pre-clinical treatment rationale

BRAF V600 mutations have been found in 1-2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50-80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decision

Las asignaturas de Álgebra Lineal y Geometría Lineal en el Grado en Matemáticas de la Universidad de Alicante

Después de varios años de implantación del Grado en Matemáticas de la Facultad de Ciencias de la Universidad de Alicante se plantea la necesidad de analizar el desarrollo de las asignaturas impartidas en el mismo y de abordar posibles mejoras. En esta red nos ocuparemos concretamente de las asignaturas correspondientes al Álgebra Lineal y a la Geometría Lineal. Estas asignaturas no han sido diseñadas por un único profesor, sino que distintos profesores han participado en la elaboración de las guías docentes y en la impartición de las mismas. Así pues, consideramos que resulta pertinente revisar de manera conjunta los contenidos para evitar solapamientos y, si procede, replantear alguno de los temas de manera que se puedan optimizar tanto los tiempos como los desarrollos teórico-prácticos de las tres asignaturas revisadas. La red está coordinada por la responsable de la asignatura Geometría lineal hasta el curso 2014-2015 y constituida por los profesores que imparten las asignaturas Álgebra Lineal I, Álgebra Lineal II y Geometría Lineal y por dos alumnos del último curso. Nuestro propósito es detectar posibles lagunas, repeticiones o deficiencias y así contribuir a la mejora de la docencia en estas asignaturas a partir de propuestas concretas

Defining Optimal Conditions for Tumor Extracellular Vesicle DNA Extraction for Mutation Profiling

(1) Background: Extracellular vesicles (EVs) have emerged as crucial players in the communication between cells in both physiological and pathological scenarios. The functions of EVs are strongly determined by their molecular content, which includes all bioactive molecules, such as proteins, lipids, RNA, and, as more recently described, double-stranded DNA. It has been shown that in oncological settings DNA associated with EVs (EV-DNA) is representative of the genome of parental cells and that it reflects the mutational status of the tumor, gaining much attention as a promising source of biomarker mutant DNA. However, one of the challenges in studies of EV-DNA is the lack of standardization of protocols for the DNA extraction from EVs, as well as ways to assess quality control, which hinders its future implementation in clinics. (2) Methods: We performed a comprehensive comparison of commonly used approaches for EV-DNA extraction by assessing DNA quantity, quality, and suitability for downstream analyses. (3) Results: We here established strategic points to consider for EV-DNA preparation for mutational analyses, including qPCR and NGS. (4) Conclusions: We put in place a workflow that can be applied for the detection of clinically relevant mutations in the EV-DNA of cancer patients

Anuario de estudios celianos 2014-15

La Universidad Camilo José Cela recoge en estos anuarios las investigaciones que se llevan a cabo cada año sobre la obra de quien fue su rector Honorario. Se compromete así, en colaboración con la Fundación que también lleva su nombre, con la herencia literaria y la memoria de CJC, y favorece la divulgación de las conclusiones de los estudios más importantes realizados cada año. El presente número doble (2014-2015) del Anuario de estudios celianos se articula excepcionalmente en dos apartados: ensayos y artículos y los anexos en los que se recogen los textos ganadores del VI y VII Premio de relatos Camilo José Cela para jóvenes. Una revista de vuelo universitario quiere estar también vinculada a la creación literaria que realizan las nuevas generacionesCátedra Camilo José Cela de Estudios Hispánico

At the beginnings of the funerary Megalithism in Iberia at Campo de Hockey necropolis

[EN] The excavations undertaken at the Campo de Hockey site in 2008 led to the identification of a major Neolithic necropolis in the former Island of San Fernando (Bay of Cadiz). This work presents the results of the latest studies, which indicate that the site stands as one of the oldest megalithic necropolises in the Iberian Peninsula. The main aim of this work is to present with precision the chronology of this necropolis through a Bayesian statistical model that confirms that the necropolis was in use from c. 4300 to 3800 cal BC. The presence of prestige grave goods in the earliest and most monumental graves suggest that the Megalithism phenomenon emerged in relation to maritime routes linked to the distribution of exotic products. We also aim to examine funerary practices in these early megalithic communities, and especially their way of life and the social reproduction system. As such, in addition to the chronological information and the Bayesian statistics, we provide the results of a comprehensive interdisciplinary study, including anthropological, archaeometric and genetic data.We wish to express our gratitude to Antonio Saez Espligares (Historical Museum of San Fernando) and Lourdes Lorenzo (Figlina, s.l.) for their support during the archaeological excavation. This research was conducted in the framework of the following research projects: "Analysis of prehistoric societies from the Middle Palaeolithic to the Late Neolithic at both sides of the Strait of Gibraltar: relations and contacts", funded by the State Research Agency (SRA) and the European Regional Development Fund (ERDF). Ref.: HAR2017-87324-P. (2018-2021). "Analisis interdisciplinar para el conocimiento del poblamiento humano de la Bahia de Cadiz durante la Prehistoria Reciente (VI-II milenios a.n.e.)", funded by 2014-2020 ERDF Operational Programme and the Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia. Ref.: FEDER-UCA18-106917 (2020-2023). "Analisis de los isotopos de oxigeno en conchas y de los isotopos estables de oxigeno y carbono en huesos humanos en el poblado neolitico insular de Campo de Hockey (San Fernando, Cadiz)", authorised and funded by CEIMAR. Ref.: CEIJ-015 (2018-2019). Eduardo Molina Piernas acknowledges co-funding from European Social Fund (D1113102E3) and Junta de Andalucia

BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer

Altres ajuts: Fellowship Award of the International Association for the Study of Lung Cancer i grant of the Italian Association for Cancer Research (AIRC My First AIRC Grant n° 14282).Abstract.BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR

Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing : results of the RING observational trial.

Abstract Several platforms for noninvasive EGFR testing are currently used in the clinical setting with sensitivities ranging from 30% to 100%. Prospective studies evaluating agreement and sources for discordant results remain lacking. Herein, seven methodologies including two next-generation sequencing (NGS)-based methods, three high-sensitivity PCR-based platforms, and two FDA-approved methods were compared using 72 plasma samples, from EGFR-mutant non-small-cell lung cancer (NSCLC) patients progressing on a first-line tyrosine kinase inhibitor (TKI). NGS platforms as well as high-sensitivity PCR-based methodologies showed excellent agreement for EGFR-sensitizing mutations (K = 0.80-0.89) and substantial agreement for T790M testing (K = 0.77 and 0.68, respectively). Mutant allele frequencies (MAFs) obtained by different quantitative methods showed an excellent reproducibility (intraclass correlation coefficients 0.86-0.98). Among other technical factors, discordant calls mostly occurred at mutant allele frequencies (MAFs) ≤ 0.5%. Agreement significantly improved when discarding samples with MAF ≤ 0.5%. EGFR mutations were detected at significantly lower MAFs in patients with brain metastases, suggesting that these patients risk for a false-positive result. Our results support the use of liquid biopsies for noninvasive EGFR testing and highlight the need to systematically report MAFs. Keywords: NGS; circulating free DNA; epidermal growth factor receptor; non-small-cell lung cancer; osimertinib; tyrosine kinase inhibitor

R-Score: A New Parameter to Assess the Quality of Variants’ Calls Assessed by NGS Using Liquid Biopsies

Next-generation sequencing (NGS) has enabled a deeper knowledge of the molecular landscape in non-small cell lung cancer (NSCLC), identifying a growing number of targetable molecular alterations in key genes. However, NGS profiling of liquid biopsies risk for false positive and false negative calls and parameters assessing the quality of NGS calls remains lacking. In this study, we have evaluated the positive percent agreement (PPA) between NGS and digital PCR calls when assessing EGFR mutation status using 85 plasma samples from 82 EGFR-positive NSCLC patients. According to our data, variant allele fraction (VAF) was significantly lower in discordant calls and the median of the absolute values of all pairwise differences (MAPD) was significantly higher in discordant calls (p < 0.001 in both cases). Based on these results, we propose a new parameter that integrates both variables, named R-score. Next, we sought to evaluate the PPA for EGFR mutation calls between two independent NGS platforms using a subset of 40 samples from the same cohort. Remarkably, there was a significant linear correlation between the PPA and the R-score (r = 0.97; p < 0.001). Specifically, the PPA of samples with an R-score ≤ −1.25 was 95.83%, whereas PPA falls to 81.63% in samples with R-score ≤ 0.25. In conclusion, R-score significantly correlates with PPA and can assist laboratory medicine specialists and data scientists to select reliable variants detected by NGS

Las asignaturas de Álgebra Lineal y Geometría Lineal en el Grado en Matemáticas de la Universidad de Alicante

Después de varios años de implantación del Grado en Matemáticas de la Facultad de Ciencias de la Universidad de Alicante se plantea la necesidad de analizar el desarrollo de las asignaturas impartidas en el mismo y de abordar posibles mejoras. En esta red nos ocuparemos concretamente de las asignaturas correspondientes al Álgebra Lineal y a la Geometría Lineal. Estas asignaturas no han sido diseñadas por un único profesor, sino que distintos profesores han participado en la elaboración de las guías docentes y en la impartición de las mismas. Así pues, consideramos que resulta pertinente revisar de manera conjunta los contenidos para evitar solapamientos y, si procede, replantear alguno de los temas de manera que se puedan optimizar tanto los tiempos como los desarrollos teórico-prácticos de las tres asignaturas revisadas. La red está coordinada por la responsable de la asignatura Geometría lineal hasta el curso 2014-2015 y constituida por los profesores que imparten las asignaturas Álgebra Lineal I, Álgebra Lineal II y Geometría Lineal y por dos alumnos del último curso. Nuestro propósito es detectar posibles lagunas, repeticiones o deficiencias y así contribuir a la mejora de la docencia en estas asignaturas a partir de propuestas concretas