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:This dataset spans two studies which investigate the clinico-pathology and molecular features of infant medulloblastoma, defined as children aged <5 years at diagnosis. Both abstracts follow. Background Infant medulloblastoma (iMB; <3-5-years old at diagnosis) is a major challenge in paediatric oncology. The favourable-risk SHH iMB molecular group with desmoplastic histology (40% of iMB) is commonly spared upfront craniospinal irradiation (CSI) to avoid neurocognitive side-effects. However, clinical studies of the remaining non-WNT/non-SHH iMB group (~60% of iMBs) have not been undertaken, and it is currently considered uniformly high-risk. Understanding the potential for its biological subclassification and clinical stratification is an essential goal towards improved outcomes. This study therefore aimed to directly compare different therapeutic approaches in non-WNT/non-SHH iMB and determine relationships between outcomes and clinico-molecular features. Methods We assembled a global cohort of non-WNT/non-SHH iMBs (n=375). Inclusion criteria was a principal non-WNT/non-SHH MB classification (Group 3 or Group 4); confidence score >0.8) using DNA methylation array-based classification. The survival cohort (n=313) was defined by availability of complete progression-free (PFS) and overall (OS) survival data. Using these cohorts, we characterised their molecular pathology, treatments received, and relationships to outcomes, principally PFS. Findings The total collected cohort (n=375) comprised 262 males and 110 females, with a median age of 3.0 (IQR 2.53-4.0) years. Molecular group 3 tumours (iMBGrp3) predominated (n=246, 66%), primarily molecular subgroups 2 (n=75; 32%), 3 (n=46; 19%) and 4 (n=98; 42%). Regimens incorporating upfront CSI were most common (54% of patients); such patients had significantly better PFS than those treated with focal or no radiotherapy (5-year PFS; CSI, 57.9%; Focal-radiotherapy, 35.3%; No radiotherapy, 26.8%; p<0.001), and equivalent PFS to CSI-treated non-infant patients. Upfront chemotherapy -only approaches were used in 33% (66/199) of iMBGrp3; high-dose (HIGH-DOSE) chemotherapy produced better survival rates (45.7%, 5-year PFS) than intraventricular methotrexate-based (21.1%, 5-year PFS) or standard-dose (11%, 2-year PFS) chemotherapy (p=0.003). Subgroup 4 represents a novel chemo-sensitive iMB group, with 64.3% 5-year PFS using high-dose -chemotherapy-only and with evidence of rescue post-relapse. MYC-amplified iMBGrp3 ( subgroup2/3) have dismal prognosis (0% 2-year overall survival (OS)), with rapid progression regardless of treatment. Remaining subgroup 2/3 patients treated with Chemotherapy-only had 30% 5-year PFS, showed some evidence of post-relapse rescue, and could be randomised to test alternative therapeutic strategies. Interpretation Non-WNT/non-SHH iMB outcomes are associated with specific biomarkers and therapy received. Capturing this ‘real-world’ experience identifies favourable ( subgroup 4) and very-high-risk ( subgroup 2/3-MYC) patient groups, providing a critical foundation for first biomarker-driven iMBGrp3 clinical trials. Background Medulloblastoma in infants (iMB; <5 years) presents the challenge of achieving cure while minimizing deleterious cranio-spinal irradiation (CSI)-associated late-effects. Non-randomised Phase-II studies have examined upfront CSI omission and chemotherapy intensification for favourable-risk desmoplastic/nodular (DN) tumours associated with the SHH molecular group (iMBSHH). Comparison of these therapies in large molecularly-defined iMBSHH cohorts, alongside investigations of prognostic biomarkers in therapy-specific context, is urgently required to define future therapeutic strategies. Methods A multi-national cohort of molecularly and clinically-annotated iMBSHH (n=267), not previously reported in clinical trials, was assembled. We investigated molecular pathology, upfront treatments, and relationships to outcome, in this ‘real-world’ setting. Findings iMBSHH patients treated upfront with intensified chemotherapy-only regimens (n=132) based on intraventricular methotrexate (IVT-MTX; 5-year progression-free survival (PFS), 72.6%) or high-dose (HD; 73.0%) therapy, achieved (PFS) outcomes comparable to upfront CSI-based regimens (n=49; 74.0%)(p=0.51). Lower-intensity standard-dose chemotherapy-only regimens were inferior (SD; 48.4% PFS)(p=0.006). Rescue was common post-relapse after IVT-MTX/HD protocols and translated into 5-year overall survival (OS) of 85.6% and 88.6%, respectively. iMBSHH encompassed SHH-1 (38%), SHH-2 (47%) and SHH-3 (14%) WHO subgroups. In CSI-naïve iMBSHH, SD associated with worse PFS in SHH-1 (p=0.001), but not SHH-2. Non-DN/MBEN histology (21.2% of iMBSHH) conferred worse PFS in upfront CSI-treated and SD (p<0.001 and p=0.003, respectively) groups. Metastatic disease only associated with prognosis with upfront IVT-MTX-only therapies (p=0.013), while established high-risk features of non-infant MBSHH (TP53-mutation, LCA-histology, MYCN-amplification) only associated with poor prognosis in older SHH-3 (7/7 relapsed). Finally, CSI-naïve PFS findings were validated in a re-evaluation of smaller historical trials cohorts. Interpretation iMBSHH outcomes and prognostic biomarkers are therapy-dependent. Non-metastatic iMBSHH treated with HD or IVT-MTX chemotherapy-only have equivalent favourable outcomes, independent of histology and subgroup. With outcomes established, clinical trials must now focus on quality-of-life following different intensified approaches, to identify kindest curative strategies.

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.

Project description:Recent genomic approaches have suggested the existence of multiple distinct subtypes of medulloblastoma. We studied a large cohort of medulloblastomas to determine how many subgroups of the disease exist, how they differ, and the extent of overlap between subgroups. We determined gene expression profiles and DNA copy number aberrations for 103 primary medulloblastomas. Bioinformatic tools were used for class discovery of medulloblastoma subgroups based on the most informative genes in the dataset. Immunohistochemistry for subgroup-specific ‘signature’ genes was used to determine subgroup affiliation for 294 non-overlapping medulloblastomas on two independent tissue microarrays (TMAs). Multiple unsupervised analyses of transcriptional profiles identified four distinct, non-overlapping molecular variants: WNT, SHH, Group C, and Group D. Supervised analysis of these four subgroups revealed significant subgroup-specific demographics, histology, metastatic status, and DNA copy number aberrations. Immunohistochemistry for DKK1 (WNT), SFRP1 (SHH), NPR3 (Group C), and KCNA1 (Group D) could reliably and uniquely classify formalin fixed medulloblastomas in ~98% of cases. Group C patients (NPR3 +ve tumors) exhibited a significantly diminished progression free and overall survival irrespective of their metastatic status. Our integrative genomics approach to a large cohort of medulloblastomas has identified four disparate subgroups with distinct demographics, clinical presentation, transcriptional profiles, genetic abnormalities, and clinical outcome. Medulloblastomas can be reliably assigned to subgroups through immunohistochemistry, thereby making medulloblastoma sub-classification widely available. Future research on medulloblastoma and the development of clinical trials should take into consideration these four distinct types of medulloblastoma. A total of 103 primary medulloblastoma specimens were profiled by Affymetrix exon array and gene-level analysis was performed.

Project description:Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype). The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype), arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem that included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer. A total of 16 samples are analyzed, repsresenting 4 experimental groups: Ctnnb1 medulloblastoma (3 samples); Ptch1 medulloblastoma (6 samples); embryonic dorsal brainstem (4 samples); and postnatal granule neuron precursor cells (3 samples). Every sample was prepared from a different mouse.

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International Phase III Medulloblastoma Trial (SJMB03)

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