Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.
Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and MethodsA combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180).Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively (P , .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. ConclusionBRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy. response to therapy and clinical outcome is still not known. As a result, as far as nonsurgical treatment is concerned, all patients with PLGGs receive similar treatment independent of their tumor's molecular alterations. 6 For deeply located tumors, such as hypothalamic/chiasmatic LGGs, the need for biopsy before treatment decisions are made for these children is still debated.The BRAF V600E mutation, which is observed in a variety of adult 7 and pediatric neoplasms, is thought to be present in only a small percentage of PLGGs.8 Controversy still exists as to whether BRAF V600E-mutant PLGG constitutes a unique subgroup with respect to natural history and outcome. 9,10 We have previously reported that PLGGs that transform to high-grade gliomas have a high incidence of BRAF V600E mutations in combination with CDKN2A deletion.11 CDKN2A is a tumor suppressor gene and a key regulator of the cell cycle. CDKN2A alterations act as a secondary hit, which allows for escape from cell cycle regulation and malignant behavior in multiple cancer types. 12,13 In PLGGs, CDKN2A loss has been reported to be associated with escape from oncogene-induced senescence, 14 especially when combined with BRAF mutations.To better define the clinical significance of BRAF V600E in these tumors, we performed a combined clinical and genetic analysis in an institutional discovery cohort of patients with PLGG who were diagnosed and treated in southern Ontario. 15 We then asse...
Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.
Purpose Vinblastine monotherapy has shown promising activity and a low-toxicity profile in patients with pediatric low-grade glioma (PLGG) who experienced treatment failure after initial treatment with chemotherapy and/or radiation. The aim of this study was to assess the activity of vinblastine in therapy-naïve children. Patients and Methods Patients < 18 years old with unresectable and/or progressive therapy-naïve PLGG were eligible. Vinblastine was administered once per week at a dose of 6 mg/m intravenously over a period of 70 weeks. Vision, quality of life, neurofibromatosis type 1 (NF1) status, and BRAF mutation/fusion status were also determined and correlated with outcome. Results Fifty-four patients were enrolled onto the study, with a median age of 8 years (range, 0.7 to 17.2 years). Most patients had chiasmatic/hypothalamic tumors (55.5%), and 13 patients (24.1%) had NF1. The most common histology was pilocytic astrocytoma (46.3%). Seventeen patients were diagnosed using radiologic criteria alone. Best response to chemotherapy was centrally reviewed with a response rate (complete, partial, or minor response) of 25.9%. Disease stabilization (complete, partial, or minor response or stable disease) was achieved in 47 patients (87.0%). Visual improvement was observed in 20% of patients with optic pathway glioma. Five-year overall survival and progression-free survival (PFS) rates were 94.4% (95% CI, 88.5% to 100%) and 53.2% (95% CI, 41.3% to 68.5%), respectively, for the entire cohort. Patients with NF1 had a significantly better PFS (85.1%; 95% CI, 68.0% to 100%) when compared with patients without NF1 (42.0%; 95% CI, 29.1% to 60.7%; P = .012). Age< 3 years or > 10 years was not associated with poor outcome. Treatment was well tolerated, and quality of life was not affected during treatment. In this trial, there was no correlation between BRAF alterations and outcome. Conclusion Vinblastine administered once per week is well tolerated in children with treatment naïve PLGG. Overall survival and PFS are comparable to current therapies, with a favorable toxicity profile and a maintained quality of life.
The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Deltaycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0353-0) contains supplementary material, which is available to authorized users.
Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.
Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.
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