Scott L. Pomeroy

Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

Prediction of central nervous system embryonal tumour outcome based on gene expression

Embryonal tumours of the central nervous system (CNS) represent a heterogeneous group of tumours about which little is known biologically, and whose diagnosis, on the basis of morphologic appearance alone, is controversial. Medulloblastomas, for example, are the most common malignant brain tumour of childhood, but their pathogenesis is unknown, their relationship to other embryonal CNS tumours is debated, and patients’ response to therapy is difficult to predict. We approached these problems by developing a classification system based on DNA microarray gene expression data derived from 99 patient samples. Here we demonstrate that medulloblastomas are molecularly distinct from other brain tumours including primitive neuroectodermal tumours (PNETs), atypical teratoid/rhabdoid tumours (AT/RTs) and malignant gliomas. Previously unrecognized evidence supporting the derivation of medulloblastomas from cerebellar granule cells through activation of the Sonic Hedgehog (SHH) pathway was also revealed. We show further that the clinical outcome of children with medulloblastomas is highly predictable on the basis of the gene expression profiles of their tumours at diagnosis.

The landscape of somatic copy-number alteration across human cancers

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.

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.

From the Cover: Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

Dissecting the genomic complexity underlying medulloblastoma

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour–normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.

Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences

Tumour cells release an abundance of microvesicles containing a selected set of proteins and RNAs. Here, we show that tumour microvesicles also carry DNA, which reflects the genetic status of the tumour, including amplification of the oncogene c-Myc. We also find amplified c-Myc in serum microvesicles from tumour-bearing mice. Further, we find remarkably high levels of retrotransposon RNA transcripts, especially for some human endogenous retroviruses, such as LINE-1 and Alu retrotransposon elements, in tumour microvesicles and these transposable elements could be transferred to normal cells. These findings expand the nucleic acid content of tumour microvesicles to include: elevated levels of specific coding and non-coding RNA and DNA, mutated and amplified oncogene sequences and transposable elements. Thus, tumour microvesicles contain a repertoire of genetic information available for horizontal gene transfer and potential use as blood biomarkers for cancer.

MEDULLOBLASTOMA EXOME SEQUENCING UNCOVERS SUBTYPE-SPECIFIC SOMATIC MUTATIONS

Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.

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.

Abnormal Cerebellar Development and Foliation in BDNF−/− Mice Reveals a Role for Neurotrophins in CNS Patterning

While target-derived neurotrophins are required for the survival of developing neurons in the PNS, the functions of neurotrophins in the CNS are unclear. Mice with a targeted gene deletion of brain-derived neurotrophic factor (BDNF) exhibit a wide-based gait. Consistent with this behavioral evidence of cerebellar dysfunction, there is increased death of granule cells, stunted growth of Purkinje cell dendrites, impaired formation of horizontal layers, and defects in the rostral-caudal foliation pattern. These abnormalities are accompanied by decreased Trk activation in granule and Purkinje cells of mutant animals, indicating that both cell types are direct targets for BDNF. These data suggest that BDNF acts as an anterograde or an autocrine-paracrine factor to regulate survival and morphologic differentiation of developing CNS neurons, and thereby affects neural patterning.