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1.
Cell ; 153(5): 1064-79, 2013 May 23.
Article in English | MEDLINE | ID: mdl-23706743

ABSTRACT

Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:


Subject(s)
Caenorhabditis elegans/metabolism , Elongation Factor 2 Kinase/metabolism , Neoplasms/physiopathology , Peptide Chain Elongation, Translational , Signal Transduction , AMP-Activated Protein Kinases/metabolism , Animals , Brain Neoplasms/physiopathology , Caenorhabditis elegans/genetics , Cell Survival , Cell Transformation, Neoplastic , Elongation Factor 2 Kinase/genetics , Food Deprivation , Glioblastoma/physiopathology , HeLa Cells , Humans , Mice , Mice, Nude , NIH 3T3 Cells , Neoplasm Transplantation , Peptide Elongation Factor 2/metabolism , Transplantation, Heterologous
2.
Nature ; 482(7386): 529-33, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22343890

ABSTRACT

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.


Subject(s)
Clonal Evolution/genetics , Medulloblastoma/genetics , Medulloblastoma/pathology , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Animals , CpG Islands/genetics , DNA Methylation , DNA Transposable Elements/genetics , Disease Models, Animal , Genes, p53/genetics , Germ-Line Mutation/genetics , Humans , Li-Fraumeni Syndrome/complications , Li-Fraumeni Syndrome/genetics , Medulloblastoma/complications , Mice , Mutagenesis, Insertional , Survival Rate
3.
Nature ; 482(7384): 226-31, 2012 Jan 29.
Article in English | MEDLINE | ID: mdl-22286061

ABSTRACT

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Glioblastoma/genetics , Histones/genetics , Mutation/genetics , Adaptor Proteins, Signal Transducing/genetics , Base Sequence , Child , Chromatin/metabolism , Co-Repressor Proteins , DNA Helicases/genetics , DNA Mutational Analysis , Exome/genetics , Gene Expression Profiling , Histones/metabolism , Humans , Molecular Chaperones , Molecular Sequence Data , Nuclear Proteins/genetics , Telomere/genetics , Tumor Suppressor Protein p53/genetics , X-linked Nuclear Protein
4.
Lancet Oncol ; 18(5): 682-694, 2017 05.
Article in English | MEDLINE | ID: mdl-28314689

ABSTRACT

BACKGROUND: The WHO classification of brain tumours describes 15 subtypes of meningioma. Nine of these subtypes are allotted to WHO grade I, and three each to grade II and grade III. Grading is based solely on histology, with an absence of molecular markers. Although the existing classification and grading approach is of prognostic value, it harbours shortcomings such as ill-defined parameters for subtypes and grading criteria prone to arbitrary judgment. In this study, we aimed for a comprehensive characterisation of the entire molecular genetic landscape of meningioma to identify biologically and clinically relevant subgroups. METHODS: In this multicentre, retrospective analysis, we investigated genome-wide DNA methylation patterns of meningiomas from ten European academic neuro-oncology centres to identify distinct methylation classes of meningiomas. The methylation classes were further characterised by DNA copy number analysis, mutational profiling, and RNA sequencing. Methylation classes were analysed for progression-free survival outcomes by the Kaplan-Meier method. The DNA methylation-based and WHO classification schema were compared using the Brier prediction score, analysed in an independent cohort with WHO grading, progression-free survival, and disease-specific survival data available, collected at the Medical University Vienna (Vienna, Austria), assessing methylation patterns with an alternative methylation chip. FINDINGS: We retrospectively collected 497 meningiomas along with 309 samples of other extra-axial skull tumours that might histologically mimic meningioma variants. Unsupervised clustering of DNA methylation data clearly segregated all meningiomas from other skull tumours. We generated genome-wide DNA methylation profiles from all 497 meningioma samples. DNA methylation profiling distinguished six distinct clinically relevant methylation classes associated with typical mutational, cytogenetic, and gene expression patterns. Compared with WHO grading, classification by individual and combined methylation classes more accurately identifies patients at high risk of disease progression in tumours with WHO grade I histology, and patients at lower risk of recurrence among WHO grade II tumours (p=0·0096) from the Brier prediction test). We validated this finding in our independent cohort of 140 patients with meningioma. INTERPRETATION: DNA methylation-based meningioma classification captures clinically more homogenous groups and has a higher power for predicting tumour recurrence and prognosis than the WHO classification. The approach presented here is potentially very useful for stratifying meningioma patients to observation-only or adjuvant treatment groups. We consider methylation-based tumour classification highly relevant for the future diagnosis and treatment of meningioma. FUNDING: German Cancer Aid, Else Kröner-Fresenius Foundation, and DKFZ/Heidelberg Institute of Personalized Oncology/Precision Oncology Program.


Subject(s)
DNA Methylation , Meningeal Neoplasms/classification , Meningeal Neoplasms/genetics , Meningioma/classification , Meningioma/genetics , Neoplasm Recurrence, Local/genetics , DNA Copy Number Variations , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Disease Progression , Disease-Free Survival , Female , Genome , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Male , Meningeal Neoplasms/pathology , Meningioma/pathology , Neoplasm Grading , Neoplasm Recurrence, Local/pathology , Neurofibromin 2/genetics , Nuclear Proteins/genetics , Proto-Oncogene Proteins c-akt/genetics , Retrospective Studies , Sequence Analysis, RNA , Smoothened Receptor/genetics , Survival Rate , Transcription Factors/genetics , Transcriptome , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics
5.
Proc Natl Acad Sci U S A ; 110(10): 4009-14, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23412337

ABSTRACT

Glioblastoma (GB) is the most common and aggressive primary brain malignancy, with poor prognosis and a lack of effective therapeutic options. Accumulating evidence suggests that intratumor heterogeneity likely is the key to understanding treatment failure. However, the extent of intratumor heterogeneity as a result of tumor evolution is still poorly understood. To address this, we developed a unique surgical multisampling scheme to collect spatially distinct tumor fragments from 11 GB patients. We present an integrated genomic analysis that uncovers extensive intratumor heterogeneity, with most patients displaying different GB subtypes within the same tumor. Moreover, we reconstructed the phylogeny of the fragments for each patient, identifying copy number alterations in EGFR and CDKN2A/B/p14ARF as early events, and aberrations in PDGFRA and PTEN as later events during cancer progression. We also characterized the clonal organization of each tumor fragment at the single-molecule level, detecting multiple coexisting cell lineages. Our results reveal the genome-wide architecture of intratumor variability in GB across multiple spatial scales and patient-specific patterns of cancer evolution, with consequences for treatment design.


Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Base Sequence , Chromosome Aberrations , DNA Copy Number Variations , DNA, Neoplasm/genetics , Disease Progression , Evolution, Molecular , Genes, erbB-1 , Genes, p16 , Humans , Phylogeny , Transcriptome
6.
Acta Neuropathol ; 129(6): 775-88, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25792358

ABSTRACT

Pilocytic astrocytomas (PAs) were recognized as a discrete clinical entity over 70 years ago. They are relatively benign (WHO grade I) and have, as a group, a 10-year survival of over 90%. Many require merely surgical removal and only very infrequently do they progress to more malignant gliomas. While most show classical morphology, they may present a spectrum of morphological patterns, and there are difficult cases that show similarities to other gliomas, some of which are malignant and require aggressive treatment. Until recently, almost nothing was known about the molecular mechanisms involved in their development. The use of high-throughput sequencing techniques interrogating the whole genome has shown that single abnormalities of the mitogen-activating protein kinase (MAPK) pathway are exclusively found in almost all cases, indicating that PA represents a one-pathway disease. The most common mechanism is a tandem duplication of a ≈2 Mb-fragment of #7q, giving rise to a fusion between two genes, resulting in a transforming fusion protein, consisting of the N-terminus of KIAA1549 and the kinase domain of BRAF. Additional infrequent fusion partners have been identified, along with other abnormalities of the MAP-K pathway, affecting tyrosine kinase growth factor receptors at the cell surface (e.g., FGFR1) as well as BRAF V600E, KRAS, and NF1 mutations among others. However, while the KIAA1549-BRAF fusion occurs in all areas, the incidence of the various other mutations identified differs in PAs that develop in different regions of the brain. Unfortunately, from a diagnostic standpoint, almost all mutations found have been reported in other brain tumor types, although some retain considerable utility. These molecular abnormalities will be reviewed, and the difficulties in their potential use in supporting a diagnosis of PA, when the histopathological findings are equivocal or in the choice of individualized therapy, will be discussed.


Subject(s)
Astrocytoma , Central Nervous System Neoplasms , Astrocytoma/genetics , Astrocytoma/pathology , Astrocytoma/physiopathology , Central Nervous System Neoplasms/genetics , Central Nervous System Neoplasms/pathology , Central Nervous System Neoplasms/physiopathology , Genetic Predisposition to Disease , Humans , Models, Molecular , Molecular Biology , Neuroimaging
7.
Acta Neuropathol ; 130(3): 419-34, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25931051

ABSTRACT

Glioblastoma (GBM) is a devastating tumor and few patients survive beyond 3 years. Defining the molecular determinants underlying long-term survival is essential for insights into tumor biology and biomarker identification. We therefore investigated homogeneously treated, IDH (wt) long-term (LTS, n = 10) and short-term survivors (STS, n = 6) by microarray transcription profiling. While there was no association of clinical parameters and molecular subtypes with long-term survival, STS tumors were characterized by differential polarization of infiltrating microglia with predominance of the M2 phenotype detectable both on the mRNA and protein level. Furthermore, transcriptional signatures of LTS and STS predicted patient outcome in a large, IDH (wt) cohort (n = 468). Interrogation of overlapping genomic alterations identified concurrent gain of chromosomes 19 and 20 as a favorable prognostic marker. The strong association of this co-gain with survival was validated by aCGH in a second, independent cohort (n = 124). Finally, FISH and gene expression data revealed gains to constitute low-amplitude, clonal events with a strong impact on transcription. In conclusion, these findings provide important insights into the manipulation of the innate immune system by particularly aggressive GBM tumors. Furthermore, we genomically characterize a previously unknown, clinically relevant subgroup of glioblastoma, which can easily be identified through modern neuropathological workup.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Chromosomes, Human, Pair 19 , Chromosomes, Human, Pair 20 , Glioblastoma/genetics , Glioblastoma/metabolism , Adult , Aged , Brain/metabolism , Brain/pathology , Brain/surgery , Brain Neoplasms/diagnosis , Brain Neoplasms/pathology , Cohort Studies , Female , Glioblastoma/diagnosis , Glioblastoma/pathology , Humans , Isocitrate Dehydrogenase/genetics , Macrophages/metabolism , Macrophages/pathology , Male , Microglia/metabolism , Microglia/pathology , Middle Aged , Prognosis , RNA, Messenger/metabolism , Survival Analysis , Survivors , Time Factors , Transcription, Genetic
8.
Genes Chromosomes Cancer ; 53(9): 750-68, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24832791

ABSTRACT

To outline further genetic mechanisms of transformation from follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL), we have performed whole genome array-CGH in 81 tumors from 60 patients [29 de novo DLBCL (dnDLBCL), 31 transformed DLBCL (tDLBCL), and 21 antecedent FL]. In 15 patients, paired tumor samples (primary FL and a subsequent tDLBCL) were available, among which three possessed more than two subsequent tumors, allowing us to follow specific genetic alterations acquired before, during, and after the transformation. Gain of 2p15-16.1 encompassing, among others, the REL, BCL11A, USP34, COMMD1, and OTX1 genes was found to be more common in the tDLBCL compared with dnDLBCL (P < 0.001). Furthermore, a high-level amplification of 2p15-16.1 was also detected in the FL stage prior to transformation, indicating its importance during the transformation event. Quantitative real-time PCR showed a higher level of amplification of REL, USP34, and COMMD1 (all involved in the NFκΒ-pathway) compared with BCL11A, which indicates that the altered genes disrupting the NFκΒ pathway may be the driver genes of transformation rather than the previously suggested BCL11A. Moreover, a 17q21.33 amplification was exclusively found in tDLBCL, never in FL (P < 0.04) or dnDLBCL, indicating an upregulation of genes of importance during the later phase of transformation. Taken together, our study demonstrates potential genomic markers for disease progression to clinically more aggressive forms. We also confirm the importance of the TP53-, CDKN2A-, and NFκΒ-pathways for the transformation from FL to DLBCL.


Subject(s)
Chromosome Aberrations , Chromosomes, Human, Pair 2/genetics , Lymphoma, Follicular/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child, Preschool , Chromosomes, Human, Pair 17/genetics , Disease Progression , Female , Genetic Markers , Humans , Lymphoma, Follicular/pathology , Lymphoma, Large B-Cell, Diffuse/pathology , Male , Middle Aged , NF-kappa B/metabolism , Signal Transduction
9.
Acta Neuropathol ; 126(2): 267-76, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23764841

ABSTRACT

Telomere lengthening is one of the key events in most cancers, and depends largely on telomerase activation. Telomerase activation is a well-known phenomenon in gliomas; however, its mechanism remains obscure. In this study, we investigated the presence of mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in a series of 546 gliomas. We found a high incidence of mutually exclusive mutations located at two hot spots, C228T and C250T, in all subtypes of gliomas (55 %). The frequency of mutation was particularly high among primary glioblastomas (70 %) and pure oligodendroglial tumors (74 %), while relatively low in diffuse astrocytomas and anaplastic astrocytomas (19 and 25 %, respectively). The expression level of TERT in tumors carrying those mutations was on average 6.1 times higher than that of wild-type tumors, indicating that the mutated promoter leads to upregulation of TERT. TERT promoter mutations were observed in almost all tumors harboring concurrent total 1p19q loss and IDH1/2 mutations (98 %). Otherwise TERT promoter mutations were mostly observed among IDH wild-type tumors. Most EGFR amplifications (92 %) were also associated with TERT promoter mutations. Our data indicate that mutation of the TERT promoter is one of the major mechanisms of telomerase activation in gliomas. The unique pattern of TERT promoter mutations in relation to other genetic alterations suggests that they play distinct roles in the pathogenesis of oligodendroglial and astrocytic tumors. Our results shed a new light on the role of telomerase activation in the development of adult gliomas.


Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Oligodendroglioma/genetics , Telomerase/genetics , Adult , Aged , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Cell Line, Tumor , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 19 , Female , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Incidence , Male , Middle Aged , Mutation , Oligodendroglioma/mortality , Oligodendroglioma/pathology , Prognosis , Promoter Regions, Genetic/genetics , Survival Analysis , Translocation, Genetic/genetics , Up-Regulation/genetics
10.
Acta Neuropathol ; 126(2): 291-301, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23660940

ABSTRACT

Pilocytic astrocytomas (PAs) are the most common brain tumors in pediatric patients and can cause significant morbidity, including chronic neurological deficiencies. They are characterized by activating alterations in the mitogen-activated protein kinase pathway, but little else is known about their development. To map the global DNA methylation profiles of these tumors, we analyzed 62 PAs and 7 normal cerebellum samples using Illumina 450K microarrays. These data revealed two subgroups of PA that separate according to tumor location (infratentorial versus supratentorial), and identified key neural developmental genes that are differentially methylated between the two groups, including NR2E1 and EN2. Integration with transcriptome microarray data highlighted significant expression differences, which were unexpectedly associated with a strong positive correlation between methylation and expression. Differentially methylated probes were often identified within the gene body and/or regions up- or downstream of the gene, rather than at the transcription start site. We also identified a large number of differentially methylated genes between cerebellar PAs and normal cerebellum, which were again enriched for developmental genes. In addition, we found a significant association between differentially methylated genes and SUZ12 binding sites, indicating potential disruption of the polycomb repressor complex 2 (PRC2). Taken together, these data suggest that PA from different locations in the brain may arise from region-specific cells of origin, and highlight the potential disruption of key developmental regulators during tumorigenesis. These findings have implications for future basic research and clinical trials, as therapeutic targets and drug sensitivity may differ according to tumor location.


Subject(s)
Astrocytoma/genetics , Brain Neoplasms/genetics , Cerebellar Neoplasms/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Neoplastic , Astrocytoma/pathology , Binding Sites/genetics , Brain Neoplasms/pathology , Cerebellar Neoplasms/pathology , Child , DNA Methylation/genetics , Gene Expression Profiling , Genes, Developmental/genetics , Humans , Neoplasm Proteins , Oligonucleotide Array Sequence Analysis , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Transcription Factors
11.
Acta Neuropathol ; 125(3): 351-8, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23404370

ABSTRACT

Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8 %) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.


Subject(s)
Kruppel-Like Transcription Factors/genetics , Meningeal Neoplasms/genetics , Meningioma/genetics , Mutation/genetics , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics , DNA Mutational Analysis , Genomics , Glutamine/genetics , Humans , Kruppel-Like Factor 4 , Lysine/genetics , Meningioma/pathology , Polymorphism, Single Nucleotide/genetics
12.
Acta Neuropathol ; 126(5): 757-62, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24096618

ABSTRACT

The activating E17K mutation in the AKT1 gene has been detected in several tumor entities. Currently several clinical studies with specific AKT1 inhibitors are under way. To determine whether AKT1 mutations are involved in human tumors of the nervous system, we examined a series of 1,437 tumors including 391 primary intracranial brain tumors and 1,046 tumors of the coverings of the central and peripheral nervous system. AKT1E17K mutations were exclusively seen in meningiomas and occurred in 65 of 958 of these tumors. A strong preponderance was seen in the variant of meningothelial meningioma WHO grade I of basal and spinal localization. In contrast, AKT1E17K mutations were rare in WHO grade II and absent in WHO grade III meningiomas. In order to more effectively detect this mutation, we tested for immunohistochemical markers associated with this alteration. We observed strong up-regulation of SFRP1 expression in all meningiomas with AKT1E17K mutation and in HEK293 cells after transfection with mutant AKT1E17K, but not in meningiomas and HEK293 cells lacking this mutation.


Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Meningeal Neoplasms/genetics , Meningioma/genetics , Mutation , Proto-Oncogene Proteins c-akt/genetics , Biomarkers, Tumor/analysis , DNA Mutational Analysis , HEK293 Cells , Humans , Immunoblotting , Immunohistochemistry , Meningeal Neoplasms/metabolism , Meningeal Neoplasms/pathology , Meningioma/metabolism , Meningioma/pathology , Neoplasm Grading , Reverse Transcriptase Polymerase Chain Reaction , Transfection
13.
Int J Cancer ; 131(5): 1104-13, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22020830

ABSTRACT

We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.


Subject(s)
Astrocytoma/genetics , CpG Islands/genetics , DNA Methylation , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Isocitrate Dehydrogenase/genetics , Mutation/genetics , Oligodendroglioma/genetics , Tumor Suppressor Proteins/genetics , Adolescent , Adult , Astrocytoma/mortality , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Child , DNA, Neoplasm/genetics , Female , Humans , Male , Middle Aged , Oligodendroglioma/mortality , Prognosis , Promoter Regions, Genetic/genetics , Real-Time Polymerase Chain Reaction , Survival Rate , Young Adult
14.
Acta Neuropathol ; 124(6): 875-81, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23161096

ABSTRACT

Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.


Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , DNA-Binding Proteins/genetics , Neoplasms, Germ Cell and Embryonal/genetics , Neuropil/metabolism , Adolescent , Brain Neoplasms/pathology , Child , Child, Preschool , Diagnosis, Differential , Gene Expression Profiling/methods , Humans , MicroRNAs/genetics , Neoplasms, Germ Cell and Embryonal/pathology , Neuroectodermal Tumors, Primitive/genetics , Neuroectodermal Tumors, Primitive/pathology , Neuropil/pathology , RNA-Binding Proteins , Rhabdoid Tumor/genetics , Rhabdoid Tumor/pathology
15.
Acta Neuropathol ; 123(4): 473-84, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22358457

ABSTRACT

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.


Subject(s)
Cerebellar Neoplasms , Chromosome Aberrations , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 3 , Medulloblastoma , Transcriptome , Adolescent , Adult , Age Distribution , Cerebellar Neoplasms/classification , Cerebellar Neoplasms/diagnosis , Cerebellar Neoplasms/genetics , Child , Child, Preschool , Cohort Studies , Cytogenetics , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/physiology , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , International Cooperation , Kv1.1 Potassium Channel/genetics , Kv1.1 Potassium Channel/metabolism , Male , Medulloblastoma/classification , Medulloblastoma/diagnosis , Medulloblastoma/genetics , Meta-Analysis as Topic , Microarray Analysis , Multivariate Analysis , Receptors, Atrial Natriuretic Factor/genetics , Receptors, Atrial Natriuretic Factor/metabolism , Retrospective Studies , Survival Analysis , Wnt Proteins/genetics , Wnt Proteins/metabolism , Young Adult
16.
J Neurooncol ; 110(1): 21-5, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22814862

ABSTRACT

Rosette-forming glioneuronal tumors (RGNT) of the fourth ventricle are rare mixed glioneuronal tumors included in the revised WHO classification of central nervous system tumors, showing partial histological similarities to pilocytic astrocytomas. To evaluate potential similarities at the molecular level between these tumors, we analysed a series of 10 RGNT for the presence of KIAA1549-BRAF fusions using interphase fluorescence in situ hybridisation. However, we found no cases showing KIAA1549-BRAF gene fusion or BRAF (V600E) mutation. Our data support the hypothesis that RGNT may represent a distinct entity among the glioneuronal tumors of the central nervous system, with molecular features different from pilocytic astrocytomas.


Subject(s)
Brain Neoplasms/genetics , Fourth Ventricle/pathology , Ganglioglioma/genetics , Oncogene Fusion/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins B-raf/genetics , Adolescent , Adult , Brain Neoplasms/pathology , Cerebral Ventricle Neoplasms/genetics , Child , Female , Ganglioglioma/pathology , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Rosette Formation
17.
Acta Neuropathol ; 121(6): 753-61, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21327941

ABSTRACT

Diffuse astrocytomas (WHO grade II) typically present as slow-growing tumours showing significant cellular differentiation, but possessing a tendency towards malignant progression. They account for ~10% of all astrocytic tumours, with a peak incidence between 30 and 40 years of age. Median survival is reported as around 6-8 years. Mutations of TP53 and IDH1 have been described as genetic hallmarks, while copy number alterations are also relatively common. However, there is some evidence to suggest that these characteristics may vary with age. Here, we present an integrated clinicopathologic, genomic and transcriptomic analysis suggesting that paediatric and adult tumours are associated with distinct genetic signatures. For example, no childhood tumour showed mutation of IDH1/2 or TP53, virtually no copy number changes were seen, and MGMT methylation was absent. In contrast, adult tumours showed IDH1/2 mutation in 94% and TP53 mutation in 69% of cases, with multiple copy number alterations per case and hypermethylation of MGMT in the majority of tumours. These differences were associated with a worse prognosis in the adult patients. The expression array data also revealed a significant difference in the expression of a number of genes putatively involved in neural stem cell maintenance and CNS development, including DLL3, HES5, BMP2, TIMP1 and BAMBI. Genes involved in DNA replication and the cell cycle were also enriched in the adult tumours, suggesting that their more aggressive behaviour may be due to derivation from a more rapidly dividing, less differentiated cell type.


Subject(s)
Astrocytoma/genetics , Brain Neoplasms/genetics , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Gene Expression Regulation, Neoplastic/genetics , Isocitrate Dehydrogenase/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Adolescent , Adult , Age Factors , Astrocytoma/pathology , Astrocytoma/physiopathology , Brain Neoplasms/pathology , Brain Neoplasms/physiopathology , Child , DNA Copy Number Variations , DNA Methylation , DNA Mutational Analysis/methods , Female , Gene Expression Profiling/methods , Humans , Male , Middle Aged , Mutation/genetics , Oligonucleotide Array Sequence Analysis/methods , Principal Component Analysis , Survival Analysis , Young Adult
18.
Acta Neuropathol ; 121(6): 763-74, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21424530

ABSTRACT

Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in KIAA1549-BRAF fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for RAF fusion genes and mutations in KRAS, NRAS, HRAS, PTPN11, BRAF and RAF1. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming BRAF gene fusions with FAM131B, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B-BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported BRAF and RAF1 fusion variants in 72% (90/125) of PA. Mutations in BRAF (8/125), KRAS (2/125) and NF1 (4/125) and the rare RAF1 gene fusions (2/125) were mutually exclusive with BRAF rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.


Subject(s)
Astrocytoma/genetics , Brain Neoplasms/genetics , MAP Kinase Signaling System/genetics , Mutation/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins B-raf/genetics , Animals , Child, Preschool , Chromosomes, Human, Pair 7/genetics , Comparative Genomic Hybridization , Female , Follow-Up Studies , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , Infant , Male , Mice , NIH 3T3 Cells , Oligonucleotide Array Sequence Analysis , Phosphorylation/genetics , Sequence Deletion , Statistics, Nonparametric , Transfection/methods
19.
Genes Chromosomes Cancer ; 48(2): 121-31, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18973140

ABSTRACT

Isodicentric 17q is the most commonly reported chromosomal abnormality in medulloblastomas. Its frequency suggests that genes disrupted in medulloblastoma formation may play a role in tumorigenesis. We have previously identified two chromosome 17 breakpoint at a 1 Mb resolution. Our aims were to accurately map the position of these breakpoints and to identify mechanisms of gene disruption at this site. CGH with a custom tiling path genomic BAC array of chromosome 17 enriched with fosmids at the breakpoint regions was used to analyze a series of 45 medulloblastomas and three medulloblastoma-derived cell lines. In total, 17 of 45 medulloblastomas had an isodicentric 17q. Two breakpoint regions were identified and their positions were mapped. The array identified a more complex arrangement at the breakpoint than has been reported previously using lower resolution BAC arrays. The patterns observed indicated that dicentric chromosome formation occurs both via nonallelic homologous recombination between palindromically arranged low copy repeats (the previously accepted mechanism) and by recombination between nonidentical sequences. In addition, novel alternative structural alterations, a homozygous deletion and a duplication, were identified within the chromosome breakpoint region in two cases. At the resolution of the array, these structural alterations spanned the same genes as cases with dicentric 17q formation, implying that the disruption of genes at the chromosome breakpoint itself may be of greater biological significance than has previously been suspected.


Subject(s)
Chromosome Breakage , Chromosomes, Human, Pair 17/genetics , Medulloblastoma/genetics , Adolescent , Adult , Child , Child, Preschool , Chromosome Mapping , Comparative Genomic Hybridization , Female , Gene Deletion , Gene Dosage , Gene Duplication , Humans , Male , Oligonucleotide Array Sequence Analysis , Recombination, Genetic
20.
Neuro Oncol ; 11(4): 341-7, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19435942

ABSTRACT

We screened exon 4 of the gene isocitrate dehydrogenase 1 (NADP+), soluble (IDH1) for mutations in 596 primary intracranial tumors of all major types. Codon 132 mutation was seen in 54% of astrocytomas and 65% of oligodendroglial tumors but in only 6% of glioblastomas (3% of primary and 50% of secondary glioblastomas). There were no mutations in any other type of tumor studied. While mutations in the tumor protein p53 gene (TP53) and total 1p/19q deletions were mutually exclusive, IDH1 mutations were strongly correlated with these genetic abnormalities. All four types of mutant IDH1 proteins showed decreased enzymatic activity. The data indicate that IDH1 mutation combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas but not in primary glioblastomas.


Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Isocitrate Dehydrogenase/genetics , Mutation/genetics , Oligodendroglioma/genetics , Adult , Biomarkers, Tumor/genetics , Brain Neoplasms/enzymology , Brain Neoplasms/pathology , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 19/genetics , Comparative Genomic Hybridization , Exons/genetics , Genotype , Glioblastoma/enzymology , Glioblastoma/pathology , Humans , Loss of Heterozygosity , Oligodendroglioma/enzymology , Oligodendroglioma/pathology , Prognosis , Tumor Suppressor Protein p53/genetics
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