Identification and Functional Analysis of a Novel CTNNB1 Mutation in Pediatric Medulloblastoma

Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3 CTNNB1 mutations and were immunostained for nuclear β-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of β-catenin Ser37 residue (ΔS37). The ΔS37 β-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type β-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 β-catenin contributed to early medulloblastoma tumorigenesis.This work was funded by Asociación Pablo Ugarte APU (BC/A/14/015), Pequerropa (BC/A/15/010), and the childhood cancer support Platform from EITB Media, SAU (BIO13/CI/016/BC). R.P. was funded by Ministerio de Economía y Competitividad (Spain and Fondo Europeo de Desarrollo Regional, grant number SAF2016-79847-R). C.E.N.-X. was funded by Instituto de Salud Carlos III (Spain and the European Social Fund+, grant number: CP20/00008). P.A.-P. was supported by a Basque Government fellowship (PRE_2020_2_0116)

Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma

Lipid imaging mass spectrometry (LIMS) has been tested in several pathological contexts, demonstrating its ability to segregate and isolate lipid signatures in complex tissues, thanks to the technique’s spatial resolution. However, it cannot yet compete with the superior identification power of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), and therefore, very often, the latter is used to refine the assignment of the species detected by LIMS. Also, it is not clear if the differences in sensitivity and spatial resolution between the two techniques lead to a similar panel of biomarkers for a given disease. Here, we explore the capabilities of LIMS and HPLC-MS to produce a panel of lipid biomarkers to screen nephrectomy samples from 40 clear cell renal cell carcinoma patients. The same set of samples was explored by both techniques, and despite the important differences between them in terms of the number of detected and identified species (148 by LIMS and 344 by HPLC-MS in negative-ion mode) and the presence/absence of image capabilities, similar conclusions were reached: using the lipid fingerprint, it is possible to set up classifiers that correctly identify the samples as either healthy or tumor samples. The spatial resolution of LIMS enables extraction of additional information, such as the existence of necrotic areas or the existence of different tumor cell populations, but such information does not seem determinant for the correct classification of the samples, or it may be somehow compensated by the higher analytical power of HPLC-MS. Similar conclusions were reached with two very different techniques, validating their use for the discovery of lipid biomarkers.The work was funded by the Basque Government (IT971-16, IT1162-19, and ELKARTEK KK2018-00090) and has been developed as a Ph.D. project of LMS, who is the recipient of a Predoctoral Fellowship from the Spanish Government (BES- 2016-078721) . The authors are grateful to SGiker Lipidomic Service (UPV/EHU, MICINN, GV/EG, ESF) for the expert advice and technical and human support in MALDI and HPLC- MS analysis

Liver osteopontin is required to prevent the progression of age-related nonalcoholic fatty liver disease

[EN] Osteopontin (OPN), a senescence-associated secretory phenotype factor, is increased in patients with nonalcoholic fatty liver disease (NAFLD). Cellular senescence has been associated with age-dependent hepatosteatosis. Thus, we investigated the role of OPN in the age-related hepatosteatosis. For this, human serum samples, animal models of aging, and cell lines in which senescence was induced were used. Metabolic fluxes, lipid, and protein concentration were determined. Among individuals with a normal liver, we observed a positive correlation between serum OPN levels and increasing age. This correlation with age, however, was absent in patients with NAFLD. In wild-type (WT) mice, serum and liver OPN were increased at 10 months old (m) along with liver p53 levels and remained elevated at 20m. Markers of liver senescence increased in association with synthesis and concentration of triglycerides (TG) in 10m OPN-deficient (KO) hepatocytes when compared to WT hepatocytes. These changes in senescence and lipid metabolism in 10m OPN-KO mice liver were associated with the decrease of 78 kDa glucose-regulated protein (GRP78), induction of ER stress, and the increase in fatty acid synthase and CD36 levels. OPN deficiency in senescent cells also diminished GRP78, the accumulation of intracellular TG, and the increase in CD36 levels. In 20m mice, OPN loss led to increased liver fibrosis. Finally, we showed that OPN expression in vitro and in vivo was regulated by p53. In conclusion, OPN deficiency leads to earlier cellular senescence, ER stress, and TG accumulation during aging. The p53-OPN axis is required to inhibit the onset of agerelated hepatosteatosis.This work was supported by Ayudas para apoyar grupos de investigación del sistema Universitario Vasco (IT971‐16 to P.A.), MINECO‐FEDER (SAF2017‐87301‐R to M.L.M‐Ch) MCIU/AEI/FEDER, UE (RTI2018‐095134‐B‐100 to P.A. and RTI2018‐099413‐B‐I00 to RN, Asociación Española contra el Cáncer, Canceres raros (M.L.M‐Ch), La Caixa Foundation (to M.L.M‐Ch), Ayudas Fundación BBVA a equipos de Investigación Científica 2018 (to M.L.M‐Ch), Xunta de Galicia (RN: 2015‐CP080 and 2016‐ PG057), Fundación BBVA (RN), and European Foundation for the Study of Diabetes (RN). ISCIII‐FEDER PI17/00535 (to C.G‐M.), ISCIII‐FEDER CP14/00181 and PI16/00823 (to A.G‐ R.), and Francisco Cobos Foundation (to A.G‐R.). CiC bioGUNE thanks MINECO for the Severo Ochoa Excellence Accreditation (SEV‐2016‐ 0644

Clinical relevance of timing of assessment of ICU mortality in patients with moderate-to-severe Acute Respiratory Distress Syndrome

Mortality is a frequently reported outcome in clinical studies of acute respiratory distress syndrome (ARDS). However, timing of mortality assessment has not been well characterized. We aimed to identify a crossing-point between cumulative survival and death in the intensive care unit (ICU) of patients with moderate-to-severe ARDS, beyond which the number of survivors would exceed the number of deaths. We hypothesized that this intersection would occur earlier in a successful clinical trial vs. observational studies of moderate/severe ARDS and predict treatment response. We conducted an ancillary study of 1580 patients with moderate-to-severe ARDS managed with lung-protective ventilation to assess the relevance and timing of measuring ICU mortality rates at different time-points during ICU stay. First, we analyzed 1303 patients from four multicenter, observational cohorts enrolling consecutive patients with moderate/severe ARDS. We assessed cumulative ICU survival from the time of moderate/severe ARDS diagnosis to ventilatory support discontinuation within 7-days, 28-days, 60-days, and at ICU discharge. Then, we compared these findings to those of a successful randomized trial of 277 moderate/severe ARDS patients. In the observational cohorts, ICU mortality (487/1303, 37.4%) and 28-day mortality (425/1102, 38.6%) were similar (p = 0.549). Cumulative proportion of ICU survivors and non-survivors crossed at day-7; after day-7, the number of ICU survivors was progressively higher compared to non-survivors. Measures of oxygenation, lung mechanics, and severity scores were different between survivors and non-survivors at each point-in-time (p < 0.001). In the trial cohort, the cumulative proportion of survivors and non-survivors in the treatment group crossed before day-3 after diagnosis of moderate/severe ARDS. In clinical ARDS studies, 28-day mortality closely approximates and may be used as a surrogate for ICU mortality. For patients with moderate-to-severe ARDS, ICU mortality assessment within the first week of a trial might be an early predictor of treatment response

Stratification for Identification of Prognostic Categories in the Acute RESpiratory Distress Syndrome (SPIRES) Score

OBJECTIVES: To develop a scoring model for stratifying patients with acute respiratory distress syndrome into risk categories (Stratification for identification of Prognostic categories In the acute RESpiratory distress syndrome score) for early prediction of death in the ICU, independent of the underlying disease and cause of death. DESIGN: A development and validation study using clinical data from four prospective, multicenter, observational cohorts. SETTING: A network of multidisciplinary ICUs. PATIENTS: One-thousand three-hundred one patients with moderate-to-severe acute respiratory distress syndrome managed with lung-protective ventilation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The study followed Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis guidelines for prediction models. We performed logistic regression analysis, bootstrapping, and internal-external validation of prediction models with variables collected within 24 hours of acute respiratory distress syndrome diagnosis in 1,000 patients for model development. Primary outcome was ICU death. The Stratification for identification of Prognostic categories In the acute RESpiratory distress syndrome score was based on patient's age, number of extrapulmonary organ failures, values of end-inspiratory plateau pressure, and ratio of Pao2to Fio2assessed at 24 hours of acute respiratory distress syndrome diagnosis. The pooled area under the receiver operating characteristic curve across internal-external validations was 0.860 (95% CI, 0.831-0.890). External validation in a new cohort of 301 acute respiratory distress syndrome patients confirmed the accuracy and robustness of the scoring model (area under the receiver operating characteristic curve = 0.870; 95% CI, 0.829-0.911). The Stratification for identification of Prognostic categories In the acute RESpiratory distress syndrome score stratified patients in three distinct prognostic classes and achieved better prediction of ICU death than ratio of Pao2to Fio2at acute respiratory distress syndrome onset or at 24 hours, Acute Physiology and Chronic Health Evaluation II score, or Sequential Organ Failure Assessment scale. CONCLUSIONS: The Stratification for identification of Prognostic categories In the acute RESpiratory distress syndrome score represents a novel strategy for early stratification of acute respiratory distress syndrome patients into prognostic categories and for selecting patients for therapeutic trials