Investigation of the molecular and clinical heterogeneity of medulloblastoma subgroups

Background Medulloblastoma is the most common paediatric malignant brain tumour, with heterogeneous clinico-molecular characteristics and survival outcomes. Current WHO classification distinguishes 3 molecular subgroups: WNT, SHH (named after characteristic activation of the WNT/wingless and Sonic Hedgehog signalling pathways) and non-WNT/non-SHH medulloblastoma. Current risk stratification incorporates molecular and clinico-pathological disease correlates. Survival is associated with high-risk factors such as metastasis, large cell/anaplastic histology, MYC/MYCN amplification and subtotal resection; the presence of one or more of these factors defines high-risk disease. High-risk patients receive more intensive therapies at the cost of severe late effects as survivors. Moreover, 20% of standard-risk patients (defined by absence of all high-risk features) will die of their disease. Tumour profiling with genome-wide DNA methylation arrays is the current gold standard for molecular classification of brain tumours. Methylation arrays are also suitable for identifying DNA copy number (CN) changes, enabling simultaneous genomic and epigenomic characterisation. Hypothesis It was hypothesised that genome-wide Illumina HumanMethylation arrays provide a robust alternative to gold-standard SNP arrays for DNA CN detection and allow for single-platform, integrated genetic and epigenetic assessment , suitable for application to DNA derived from fresh-frozen and formalin-fixed, paraffin embedded tumour materials Aims Confirm usability of methylation arrays and develop methods to detect genomic alterations (aneuploidy and focal oncogene amplifications) Showcase application of methylation arrays as a cost-effective single-platform, integrated approach for improved prognostication within medulloblastoma patients. Methods In this project, methods to detect genomic alterations (aneuploidy and focal oncogene amplifications) using Illumina 450k methylation arrays were developed and validated. These methods were implemented to assess previously published cytogenetic prognostication schemes in medulloblastoma. Next, the GLMnet algorithm was used to identify prognostic methylation loci. These markers were assessed in non-WNT/non-SHH high-risk medulloblastomas and validated in an independent, mixed-risk non-WNT/non-SHH cohort. The previously published cytogenetic prognostic signature for standard-risk, non-WNT/non- SHH medulloblastoma, identified in the PNET4 clinical trial, and its potential for prognostication was assessed in high-risk, non-WNT/non-SHH disease, alone and in conjunction with methylation markers Results Easy to use methods with a low barrier to entry were developed to robustly identify genomic copy number and oncogene amplification. These methods were applied to validate two independent, previously published cytogenetic prognostication schemes within medulloblastoma. Two DNA methylation loci, mapping to MYO7A and TRIM72 genes, were identified as independently prognostic markers. A novel prognostication scheme, that combined DNA methylation markers with the PNET4 cytogenetic signature, was devised for non-WNT/non-SHH medulloblastoma. This scheme outperformed the PNET4 signature in the high-risk cohort, reclassifying 21% of high-risk patients to a favourable-risk category. Conclusion These results demonstrate the potential for routine cytogenetic assessment concurrent with molecular sub-classification using DNA methylation microarrays. Additionally, the integrated genetic and epigenetic stratification from a single platform enabled a more refined prognostication and the identification of a subset of patients, currently classified as high risk, who demonstrate improved outcomes and who may be eligible for reduced intensity treatments that would offer a better quality of life as brain tumour survivors

Citation: Epigenetic landscape correlates with genetic subtype but does not predict outcome in childhood acute lymphoblastic leukemia

Epigenetic landscape correlates with genetic subtype but does not predict outcome in childhood acute lymphoblastic leukemia. Epigenetics, 10 (8 Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University's research output. Copyright © and moral rights for items on NRL are retained by the individual author(s) and/or other copyright owners. Single copies of full items can be reproduced, displayed or performed, and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided the authors, title and full bibliographic details are given, as well as a hyperlink and/or URL to the original metadata page. The content must not be changed in any way. Full items must not be sold commercially in any format or medium without formal permission of the copyright holder. The full policy is available online: http://nrl.northumbria.ac.uk/policies.html This document may differ from the final, published version of the research and has been made available online in accordance with publisher policies. To read and/or cite from the published version of the research, please visit the publisher's website (a subscription may be required.) Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20) (p11-13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving longterm remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes

Activation of Ras and Rho GTPases and MAP Kinases by G-Protein-Coupled Receptors

14 páginas, 2 figuras.A complex intracellular signaling network mediates the multiple biological activities of G-protein-coupled receptors (GPCRs). Among them, monomeric GTPases and a family of closely related proline-targeted serine–threonine kinases, collectively known as Mitogen-Activated Protein Kinases (MAPKs), appears to play central roles in orchestrating the proliferative responses to multiple mitogens that act on GPCRs. Upon GDP/GTP exchange, monomeric GTPases control the phosphorylation of conserved threonine and tyrosine residues in MAPKs by their immediate upstream kinases, increasing their enzymatic activity and inducing their translocation to the nucleus where they phosphorylate transcription factors, thereby regulating the expression of genes playing a key role in normal and aberrant cell growth. Recently, a number of GPCRs have been engineered to provide exclusive activation by synthetic drug-like compounds while becoming insensitive to endogenous ligands. These engineered receptors, named Receptors Activated Solely by Synthetic Ligands (RASSLs), promise better understanding of GPCRs signaling in vitro and in vivo, thus representing ideal tools to selectively modulate MAPK signaling routes controlling a wide range of biological functions, from proliferation to differentiation, migration, invasion, and cell survival or death by apoptosis.Peer reviewe

Epigenetic landscape correlates with genetic subtype but does not predict outcome in childhood acute lymphoblastic leukemia.

Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20)(p11–13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving long-term remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes