Microarray-based DNA methylation profiling of medulloblastoma and normal cerebellum samples
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ABSTRACT: Robust molecular subgrouping and copy-number profiling of medulloblastoma from small amounts of archival tumor material. Validation of patterns identified by whole-genome bisulphite sequencing in a larger cohort. DNA methylation profiles of 276 primary medulloblastoma and 8 normal cerebellum control samples were generated from fresh-frozen and formalin-fixed paraffin-embedded material using the Illumina 450k methylation array.
Project description:Robust molecular subgrouping and copy-number profiling of medulloblastoma from small amounts of archival tumor material. Validation of patterns identified by whole-genome bisulphite sequencing in a larger cohort.
Project description:Here we present the case of adult medulloblastoma with minimal chromosomal rearrangements but Shh expression profile and IDH1 mutation. Data presented here are NanoString analyses of two biopsies of adult patient with a medulloblastoma which was classified as a Shh subtype Analyses were performed on 2 archival specimens of medulloblastoma, WHO Grade IV, seen at the Department of Pathology, Massachusetts General Hospital in 2007.
Project description:Purpose Integrated genomics approaches have identified at least four distinct biological variants in medulloblastoma: WNT, SHH, group C, and group D. Non-WNT/Non-SHH tumors are associated with metastatic dissemination and an unfavorable prognosis. Additional markers may enhance outcome prediction in Non-WNT/Non-SHH medulloblastomas. Experimental Design We combined transcriptomic and DNA copy-number analyses for 64 primary medulloblastomas. Bioinformatic tools were applied to discover marker genes of molecular variants. Differentially expressed transcripts were evaluated for prognostic value in the screening cohort. Immunopositivity for FSTL5 was correlated with molecular and prognostic subgroups for 235 non-overlapping medulloblastoma samples on two independent tissue microarrays (TMA). Results Unsupervised clustering analyses of transcriptome profiles confirmed four distinct molecular variants. Stable subgroup separation was achieved using only the 300 most varying transcripts. Specific distributions of clinical and molecular characteristics were noted for each cluster. Distinct expression patterns of FSTL5 in each molecular subgroup were confirmed by quantitative real-time PCR. Immunopositivity of FSTL5 identified a large cohort of patients (84 of 235 patients; 36%) at high risk for relapse and death. Importantly, over 50% of Non-WNT/Non-SHH tumors displayed FSTL5 negativity, delineating a large patient cohort with an excellent prognosis who would be considered intermediate/high-risk based on current molecular subtyping. Conclusions Comprehensive analyses of transcriptomic and genetic alterations delineate four distinct variants of medulloblastoma. The addition of FSTL5 immunohistochemistry to existing molecular stratification schemes can effectively identify those Non-WNT/Non-SHH tumors with a poor outcome. Immunohistochemical staining for FSTL5 could be a high-quality and practical tool for stratification and prognostication in future clinical trials of medulloblastoma. Whole-genome transcriptional profiling of human medulloblastomas. Subgrouping based on mRNA expression profiles. Fresh frozen tumor material was collected during tumor resection. Dye-swap design used for expression profiling. Reference was a pool of normal cerebellum tissue from 24 donors. Gene expression profiles illustrate distinct expression pattern at diagnosis. This submission represents the gene expression component of the study.
Project description:In this study, we report the first examination of Medulloblastoma (MB) methylation patterns in Arab world, using an extensive primary tumor cohort in tertiray care center. We assessed DNA methylation patterns to sub-classify clinical MB cases and their amenability to clinical applications from archival biopsy material (FPPE). We herein establish methylation events as clinically useful biomarkers and demonstrate how their incorporation into current risk-stratification schemes could significantly improve the accuracy of survival predictions in clinical setting. This holds potential for future precision therapeutic approaches aimed at improving the outcomes of afflicted MB patients.
Project description:Smoothened (SMO)-inhibitors recently entered clinical trials for sonic-hedgehog driven medulloblastoma (SHH-MB). Clinical response appears highly variable. To understand the mechanism(s) of primary resistance and to identify pathways co-operating with aberrant SHH-signaling, we sequenced a large cohort of SHH-MBs across all age groups by sequencing, DNA methylation and expression profiling. Our data show that most adults but only half of the pediatric patients with SHH-MB will respond to SMO inhibition as predicted by molecular analysis of the primary tumor and tested in the SHH-xenografts, demonstrating that the next generation of SMO-inhibitor trials should be based on these predictive biomarkers. To further dissect the biological differences between the different age groups within SHH medulloblastomas, we looked at the transcriptomic profiles of SHH medulloblastoma samples. 73 medulloblastoma samples from patients of various ages were selected for RNA extraction and hybridization on Affymetrix Human Genome U133 Plus 2.0 Arrays.
Project description:Here we present the case of adult medulloblastoma with minimal chromosomal rearrangements. Data presented here are aCGH analyses of two biopsies of adult patient with a medulloblastoma which was classified as a Shh subgroup. Analyses were performed on 2 archival specimens of medulloblastoma, WHO Grade IV, seen at the Department of Pathology, Massachusetts General Hospital from 2007. This Series represents the CGH part of the study.
Project description:Sonic hedgehog (Shh) signaling plays a critical role in regulating cerebellum development by maintaining the physiological proliferation of granule neuron precursors (GNPs), and its dysregulation leads to the oncogenesis of medulloblastoma. O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuitry. Here, we demonstrate that O-GlcNAc transferase (OGT) in GNPs mediate the cerebellum development, and the progression of the Shh-subgroup of medulloblastoma. Specifically, OGT regulates the proliferation of GNPs by activating the Shh signaling pathway via O-GlcNAcylation at S355 of GLI family zinc finger 2 (Gli2), which in turn promotes its deacetylation and transcriptional activity via dissociation from p300, a histone acetyltransferases (HATs). Inhibition of OGT via genetic ablation or chemical inhibition improves survival in a medulloblastoma mouse model. These data uncover a critical role for O-GlcNAc signaling in cerebellar development, and pinpoint a potential therapeutic target for Shh-associated medulloblastoma.
Project description:Background: Medulloblastoma (MB) is one of the most prevalent embryonal malignant brain tumors. Current classification organizes these tumors into four molecular groups (WNT-activated, SHH-activated and TP53-wild type, SHH-activated and TP53-mutant, and non-WNT/non-SHH). Recently, a comprehensive classification has been established, identifying numerous subgroups, some of which exhibit a poor prognosis. It is critical to establish effective subgrouping methods for accurate diagnosis and patient’s management that strikes a delicate balance between improving outcomes and minimizing the risk of comorbidities. Methods: We evaluated the ability of Nanopore sequencing to provide clinically relevant methylation and copy number profiles of MB. Nanopore sequencing was applied to an EPIC discovery cohort of frozen MB, benchmarked against the gold standard EPIC array, and validated further evaluated on an integrated diagnosis cohort of MB.