Richard J. Gilbertson

Molecular subgroups of medulloblastoma: the current consensus

Medulloblastoma, a small blue cell malignancy of the cerebellum, is a major cause of morbidity and mortality in pediatric oncology. Current mechanisms for clinical prognostication and stratification include clinical factors (age, presence of metastases, and extent of resection) as well as histological subgrouping (classic, desmoplastic, and large cell/anaplastic histology). Transcriptional profiling studies of medulloblastoma cohorts from several research groups around the globe have suggested the existence of multiple distinct molecular subgroups that differ in their demographics, transcriptomes, somatic genetic events, and clinical outcomes. Variations in the number, composition, and nature of the subgroups between studies brought about a consensus conference in Boston in the fall of 2010. Discussants at the conference came to a consensus that the evidence supported the existence of four main subgroups of medulloblastoma (Wnt, Shh, Group 3, and Group 4). Participants outlined the demographic, transcriptional, genetic, and clinical differences between the four subgroups. While it is anticipated that the molecular classification of medulloblastoma will continue to evolve and diversify in the future as larger cohorts are studied at greater depth, herein we outline the current consensus nomenclature, and the differences between the medulloblastoma subgroups.

Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas

To identify somatic mutations in pediatric diffuse intrinsic pontine glioma (DIPG), we performed whole-genome sequencing of DNA from seven DIPGs and matched germline tissue and targeted sequencing of an additional 43 DIPGs and 36 non-brainstem pediatric glioblastomas (non-BS-PGs). We found that 78% of DIPGs and 22% of non-BS-PGs contained a mutation in H3F3A, encoding histone H3.3, or in the related HIST1H3B, encoding histone H3.1, that caused a p.Lys27Met amino acid substitution in each protein. An additional 14% of non-BS-PGs had somatic mutations in H3F3A causing a p.Gly34Arg alteration.

Risk-adapted craniospinal radiotherapy followed by high-dose chemotherapy and stem-cell rescue in children with newly diagnosed medulloblastoma (St Jude Medulloblastoma-96): long-term results from a prospective, multicentre trial

BACKGROUND Current treatment for medulloblastoma, which includes postoperative radiotherapy and 1 year of chemotherapy, does not cure many children with high-risk disease. We aimed to investigate the effectiveness of risk-adapted radiotherapy followed by a shortened period of dose-intense chemotherapy in children with medulloblastoma. METHODS After resection, patients were classified as having average-risk medulloblastoma (< or = 1.5 cm2 residual tumour and no metastatic disease) or high-risk medulloblastoma (> 1.5 cm2 residual disease or metastatic disease localised to neuraxis) medulloblastoma. All patients received risk-adapted craniospinal radiotherapy (23.4 Gy for average-risk disease and 36.0-39.6 Gy for high-risk disease) followed by four cycles of cyclophosphamide-based, dose-intensive chemotherapy. Patients were assessed regularly for disease status and treatment side-effects. The primary endpoint was 5-year event-free survival; we also measured overall survival. This study is registered with ClinicalTrials.gov, number NCT00003211. FINDINGS Of 134 children with medulloblastoma who underwent treatment (86 average-risk, 48 high-risk), 119 (89%) completed the planned protocol. No treatment-related deaths occurred. 5-year overall survival was 85% (95% CI 75-94) in patients in the average-risk group and 70% (54-84) in those in the high-risk group (p=0.04); 5-year event-free survival was 83% (73-93) and 70% (55-85), respectively (p=0.046). For the 116 patients whose histology was reviewed centrally, histological subtype correlated with 5-year event-free survival (p=0.04): 84% (74-95) for classic histology, 77% (49-100) for desmoplastic tumours, and 57% (33-80) for large-cell anaplastic tumours. INTERPRETATION Risk-adapted radiotherapy followed by a shortened schedule of dose-intensive chemotherapy can be used to improve the outcome of patients with high-risk medulloblastoma.

Making a tumour's bed: glioblastoma stem cells and the vascular niche

Parallel to the role that normal stem cells play in organogenesis, cancer stem cells are thought to be crucial for tumorigenesis. Understanding normal development might therefore lead to better treatments of cancer. We review recent data that stem cells of glioblastoma, a highly malignant brain tumour, seem to be dependent on cues from aberrant vascular niches that mimic the normal neural stem cell niche. These data have direct implications for cancer, highlighting the similarity between normal and malignant stem cells and identifying the tumour microenvironment as a target for new therapies.

Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation

Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1C-L), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1+/C-L mice containing the Rosa26-YFP reporter allele showed that Prom1+ cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1+/C-L mice containing a Cre-dependent mutant allele of β-catenin (Ctnnb1lox(ex3)) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1+ cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1+ cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1+ tumour cells.

Genomics Identifies Medulloblastoma Subgroups That Are Enriched for Specific Genetic Alterations

PURPOSE Traditional genetic approaches to identify gene mutations in cancer are expensive and laborious. Nonetheless, if we are to avoid rejecting effective molecular targeted therapies, we must test these drugs in patients whose tumors harbor mutations in the drug target. We hypothesized that gene expression profiling might be a more rapid and cost-effective method of identifying tumors that contain specific genetic abnormalities. MATERIALS AND METHODS Gene expression profiles of 46 samples of medulloblastoma were generated using the U133av2 Affymetrix oligonucleotide array and validated using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Genetic abnormalities were confirmed using fluorescence in situ hybridization (FISH) and direct sequencing. RESULTS Unsupervised analysis of gene expression profiles partitioned medulloblastomas into five distinct subgroups (subgroups A to E). Gene expression signatures that distinguished these subgroups predicted the presence of key molecular alterations that we subsequently confirmed by gene sequence analysis and FISH. Subgroup-specific abnormalities included mutations in the Wingless (WNT) pathway and deletion of chromosome 6 (subgroup B) and mutations in the Sonic Hedgehog (SHH) pathway (subgroup D). Real-time RT-PCR analysis of gene expression profiles was then used to predict accurately the presence of mutations in the WNT and SHH pathways in a separate group of 31 medulloblastomas. CONCLUSION Genome-wide expression profiles can partition large tumor cohorts into subgroups that are enriched for specific genetic alterations. This approach may assist ultimately in the selection of patients for future clinical trials of molecular targeted therapies.

Subtypes of medulloblastoma have distinct developmental origins.

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumour subtypes that together comprise the most common malignant childhood brain tumour. These tumours are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) after 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 tumours infiltrate the dorsal brainstem, whereas SHH-subtype tumours 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 which included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice; moreover, in 15% of cases in which Tp53 was concurrently deleted, they progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence, to our knowledge, 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.

Novel mutations target distinct subgroups of medulloblastoma

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

Integrative Genomic Analysis of Medulloblastoma Identifies a Molecular Subgroup That Drives Poor Clinical Outcome

PURPOSE Medulloblastomas are heterogeneous tumors that collectively represent the most common malignant brain tumor in children. To understand the molecular characteristics underlying their heterogeneity and to identify whether such characteristics represent risk factors for patients with this disease, we performed an integrated genomic analysis of a large series of primary tumors. PATIENTS AND METHODS We profiled the mRNA transcriptome of 194 medulloblastomas and performed high-density single nucleotide polymorphism array and miRNA analysis on 115 and 98 of these, respectively. Non-negative matrix factorization-based clustering of mRNA expression data was used to identify molecular subgroups of medulloblastoma; DNA copy number, miRNA profiles, and clinical outcomes were analyzed for each. We additionally validated our findings in three previously published independent medulloblastoma data sets. RESULTS Identified are six molecular subgroups of medulloblastoma, each with a unique combination of numerical and structural chromosomal aberrations that globally influence mRNA and miRNA expression. We reveal the relative contribution of each subgroup to clinical outcome as a whole and show that a previously unidentified molecular subgroup, characterized genetically by c-MYC copy number gains and transcriptionally by enrichment of photoreceptor pathways and increased miR-183∼96∼182 expression, is associated with significantly lower rates of event-free and overall survivals. CONCLUSION Our results detail the complex genomic heterogeneity of medulloblastomas and identify a previously unrecognized molecular subgroup with poor clinical outcome for which more effective therapeutic strategies should be developed.

The brain tumor microenvironment

High‐grade brain tumors are heterogeneous with respect to the composition of bona fide tumors cells and with respect to a range of intermingling parenchymal cells. Glioblastomas harbor multiple cell types, some with increased tumorigenicity and stem cell‐like capacity. The stem‐like cells may be the cells of origin for tumor relapse. However, the tumor‐associated parenchymal cells—such as vascular cells, microglia, peripheral immune cells, and neural precursor cells—also play a vital role in controlling the course of pathology. In this review, we describe the multiple interactions of bulk glioma cells and glioma stem cells with parenchymal cell populations and highlight the pathological impact and signaling pathways known for these types of cell–cell communication. The tumor‐vasculature not only nourishes glioblastomas, but also provides a specialized niche for these stem‐like cells. In addition, microglial cells, which can contribute up to 30% of a brain tumor mass, play a role in glioblastoma cell invasion. Moreover, non‐neoplastic astrocytes can be converted into a reactive phenotype by the glioma microenvironment and can then secrete a number of factors which influences tumor biology. The young brain may have the capacity to inhibit gliomagenesis by the endogenous neural stem and progenitor cells, which secrete tumor suppressive factors. The factors, pathways, and interactions described in this review provide a new prospective on the cell biology of primary brain tumors, which may ultimately generate new treatment modalities. However, our picture of the multiple interactions between parenchymal and tumor cells is still incomplete.