Rapid, reliable, and reproducible molecular sub-grouping of clinical medulloblastoma samples

The diagnosis of medulloblastoma likely encompasses several distinct entities, with recent evidence for the existence of at least four unique molecular subgroups that exhibit distinct genetic, transcriptional, demographic, and clinical features. Assignment of molecular subgroup through routine profiling of high-quality RNA on expression microarrays is likely impractical in the clinical setting. The planning and execution of medulloblastoma clinical trials that stratify by subgroup, or which are targeted to a specific subgroup requires technologies that can be economically, rapidly, reliably, and reproducibly applied to formalin-fixed paraffin embedded (FFPE) specimens. In the current study, we have developed an assay that accurately measures the expression level of 22 medulloblastoma subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology. Comparison of the nanoString assay with Affymetrix expression array data on a training series of 101 medulloblastomas of known subgroup demonstrated a high concordance (Pearson correlation r = 0.86). The assay was validated on a second set of 130 non-overlapping medulloblastomas of known subgroup, correctly assigning 98% (127/130) of tumors to the appropriate subgroup. Reproducibility was demonstrated by repeating the assay in three independent laboratories in Canada, the United States, and Switzerland. Finally, the nanoString assay could confidently predict subgroup in 88% of recent FFPE cases, of which 100% had accurate subgroup assignment. We present an assay based on nanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials

Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas

Medulloblastoma is the most common malignant brain tumor in childhood. Molecular studies from several groups around the world demonstrated that medulloblastoma is not one disease but comprises a collection of distinct molecular subgroups. However, all these studies reported on different numbers of subgroups. The current consensus is that there are only four core subgroups, which should be termed WNT, SHH, Group 3 and Group 4. Based on this, we performed a meta-analysis of all molecular and clinical data of 550 medulloblastomas brought together from seven independent studies. All cases were analyzed by gene expression profiling and for most cases SNP or array-CGH data were available. Data are presented for all medulloblastomas together and for each subgroup separately. For validation purposes, we compared the results of this meta-analysis with another large medulloblastoma cohort (n = 402) for which subgroup information was obtained by immunohistochemistry. Results from both cohorts are highly similar and show how distinct the molecular subtypes are with respect to their transcriptome, DNA copy-number aberrations, demographics, and survival. Results from these analyses will form the basis for prospective multi-center studies and will have an impact on how the different subgroups of medulloblastoma will be treated in the future

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

Aberrant Otx2 Expression Enhances Migration and Induces Ectopic Proliferation of Hindbrain Neuronal Progenitor Cells

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation

JISTIC: Identification of Significant Targets in Cancer

<p>Abstract</p> <p>Background</p> <p>Cancer is caused through a multistep process, in which a succession of genetic changes, each conferring a competitive advantage for growth and proliferation, leads to the progressive conversion of normal human cells into malignant cancer cells. Interrogation of cancer genomes holds the promise of understanding this process, thus revolutionizing cancer research and treatment. As datasets measuring copy number aberrations in tumors accumulate, a major challenge has become to distinguish between those mutations that drive the cancer versus those passenger mutations that have no effect.</p> <p>Results</p> <p>We present JISTIC, a tool for analyzing datasets of genome-wide copy number variation to identify driver aberrations in cancer. JISTIC is an improvement over the widely used GISTIC algorithm. We compared the performance of JISTIC versus GISTIC on a dataset of glioblastoma copy number variation, JISTIC finds 173 significant regions, whereas GISTIC only finds 103 significant regions. Importantly, the additional regions detected by JISTIC are enriched for oncogenes and genes involved in cell-cycle and proliferation.</p> <p>Conclusions</p> <p>JISTIC is an easy-to-install platform independent implementation of GISTIC that outperforms the original algorithm detecting more relevant candidate genes and regions. The software and documentation are freely available and can be found at: <url>http://www.c2b2.columbia.edu/danapeerlab/html/software.html</url></p

Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma

Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors

Widespread Contribution of Gdf7 Lineage to Cerebellar Cell Types and Implications for Hedgehog-Driven Medulloblastoma Formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling

Molecular markers and potential therapeutic targets in non-WNT/non-SHH (group 3 and group 4) medulloblastomas

Childhood medulloblastomas (MB) are heterogeneous and are divided into four molecular subgroups. The provisional non-wingless-activated (WNT)/non-sonic hedgehog-activated (SHH) category combining group 3 and group 4 represents over two thirds of all MBs, coupled with the highest rates of metastases and least understood pathology. The molecular era expanded our knowledge about molecular aberrations involved in MB tumorigenesis, and here, we review processes leading to non-WNT/non-SHH MB formations.The heterogeneous group 3 and group 4 MBs frequently harbor rare individual genetic alterations, yet the emerging profiles suggest that infrequent events converge on common, potentially targetable signaling pathways. A mutual theme is the altered epigenetic regulation, and in vitro approaches targeting epigenetic machinery are promising. Growing evidence indicates the presence of an intermediate, mixed signature group along group 3 and group 4, and future clarifications are imperative for concordant classification, as misidentifying patient samples has serious implications for therapy and clinical trials.To subdue the high MB mortality, we need to discern mechanisms of disease spread and recurrence. Current preclinical models do not represent the full scale of group 3 and group 4 heterogeneity: all of existing group 3 cell lines are MYC-amplified and most mouse models resemble MYC-activated MBs. Clinical samples provide a wealth of information about the genetic divergence between primary tumors and metastatic clones, but recurrent MBs are rarely resected. Molecularly stratified treatment options are limited, and targeted therapies are still in preclinical development. Attacking these aggressive tumors at multiple frontiers will be needed to improve stagnant survival rates