Inhibition of mTOR improves malnutrition induced hepatic metabolic dysfunction.

Severe malnutrition accounts for half-a-million deaths annually in children under the age of five. Despite improved WHO guidelines, inpatient mortality remains high and is associated with metabolic dysfunction. Previous studies suggest a correlation between hepatic metabolic dysfunction and impaired autophagy. We aimed to determine the role of mTORC1 inhibition in a murine model of malnutrition-induced hepatic dysfunction. Wild type weanling C57/B6 mice were fed a 18 or 1% protein diet for two weeks. A third low-protein group received daily rapamycin injections, an mTORC1 inhibitor. Hepatic metabolic function was assessed by histology, immunofluorescence, gene expression, metabolomics and protein levels. Low protein-fed mice manifested characteristics of severe malnutrition, including weight loss, hypoalbuminemia, hypoglycemia, hepatic steatosis and cholestasis. Low protein-fed mice had fewer mitochondria and showed signs of impaired mitochondrial function. Rapamycin prevented hepatic steatosis, restored ATP levels and fasted plasma glucose levels compared to untreated mice. This correlated with increased content of LC3-II, and decreased content mitochondrial damage marker, PINK1. We demonstrate that hepatic steatosis and disturbed mitochondrial function in a murine model of severe malnutrition can be partially prevented through inhibition of mTORC1. These findings suggest that stimulation of autophagy could be a novel approach to improve metabolic function in severely malnourished children.

Multiplex Detection of Pediatric Low-Grade Glioma Signature Fusion Transcripts and Duplications Using the NanoString nCounter System.

Previous studies identified recurrent fusion and duplication events in pediatric low-grade glioma (pLGG). In addition to their role in diagnosis, the presence of these events aid in dictating therapy and predicting patient survival. Clinically, BRAF alterations are most commonly identified using fluorescent in situ hybridization (FISH). However, this method is costly, labor-intensive and does not identify nonBRAF events. Here, we evaluated the NanoString nCounter gene expression system for detecting 32 of the most commonly reported fusion/duplication events in pLGG. The assay was validated on 90 pLGG samples using FISH as the gold standard and showed sensitivity and specificity of 97% and 98%, respectively. We next profiled formalin-fixed paraffin-embedded preserved biopsy specimens from 429 pLGG cases. 171 (40%) of the cases within our cohort tested positive for a fusion or duplication event contained within our panel. These events, in order of prevalence, were KIAA1549-BRAF 16;9 (89/171, 52.0%), KIAA1549-BRAF 15;9 (42/171, 24.6%), KIAA1549-BRAF 16;11 (14/171, 8.2%), FGFR1-TACC1 17;7 (13/171, 7.6%), MYBL1 duplication (5/171, 2.9%), KIAA1549-BRAF 18;10 (4/171, 2.3%), KIAA1549-BRAF 15;11 (2/171, 1.2%), FAM131B-BRAF 2;9 (1/171, 0.6%), and RNF130-BRAF 3;9 (1/171, 0.6%). This work introduces NanoString as a viable clinical replacement for the detection of fusion and duplication events in pLGG.

Targeted detection of genetic alterations reveal the prognostic impact of H3K27M and MAPK pathway aberrations in paediatric thalamic glioma.

Paediatric brain tumours arising in the thalamus present significant diagnostic and therapeutic challenges to physicians due to their sensitive midline location. As such, genetic analysis for biomarkers to aid in the diagnosis, prognosis and treatment of these tumours is needed. Here, we identified 64 thalamic gliomas with clinical follow-up and characterized targeted genomic alterations using newly optimized droplet digital and NanoString-based assays. The median age at diagnosis was 9.25 years (range, 0.63-17.55) and median survival was 6.43 (range, 0.01-27.63) years. Our cohort contained 42 and 22 tumours reviewed as low and high grade gliomas, respectively. Five (12 %) low grade and 11 (50 %) high grade gliomas were positive for the H3F3A/HIST1H3B K27M (H3K27M) mutation. Kaplan-Meier survival analysis revealed significantly worse overall survival for patients harbouring the H3K27M mutation versus H3F3A/HIST1H3B wild type (H3WT) samples (log-rank p < 0.0001) with a median survival of 1.02 vs. 9.12 years. Mitogen-activated protein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations or fusion events were detected in 44 % of patients, and was associated with long-term survival in the absence of H3K27M (log-rank p < 0.0001). Multivariate analysis demonstrated H3K27M status and high grade histology to be the most significant independent predictors of poor overall survival with hazard ratios of 6.945 and 7.721 (p < 0.0001), respectively. In contrast, MAPK pathway activation is a predictor of favourable patient outcome, although not independent of other clinical factors. Importantly, we show that low grade malignancies may harbour H3K27M mutations and that these tumours show a dismal survival compared to low grade H3WT cases. Our data strongly supports the inclusion of targeted genetic testing in childhood thalamic tumours to most accurately stratify patients into appropriate risk groups.