Deletions of chromosome arm 10q are found in most glioblastomas and subsets of lower grade gliomas. Mutations in the PTEN gene at 10q23.3 are restricted to less than half of the 10q‐deleted gliomas, ...suggesting additional glioma‐associated tumor suppressors on 10q. We investigated 64 astrocytic gliomas of different malignancy grades for aberrant expression of 16 microRNAs (miRNAs) on 10q. Thereby, we identified four miRNAs (miR‐107, miR‐146b‐5p, miR‐346, miR‐1287‐5p) whose expression was frequently down‐regulated in anaplastic astrocytomas and/or glioblastomas. DNA methylation analyses revealed 5′‐CpG site hypermethylation of miR‐346 in more than two‐thirds of primary glioblastomas, while aberrant 5′‐CpG site methylation of miR‐146b‐5p was frequent in IDH1‐mutant astrocytomas and secondary glioblastomas. Overexpression of either of the four miRNAs in glioma cell lines reduced cell proliferation and/or increased caspase‐3/7 activity. Expression analyses of miRNA overexpressing glioma cells and 3′‐untranslated region luciferase reporter gene assays revealed evidence that these miRNAs post‐transcriptionally regulate expression of glioma‐relevant genes, including CDK6 (miR‐107), EGFR (miR‐146b‐5p, miR‐1287‐5p), TERT and SEMA6A (miR‐346), all of which are overexpressed in malignant gliomas in situ. In summary, we show that the 10q‐located miRNAs miR‐107, miR‐146b‐5p, miR‐346 and miR‐1287‐5p are frequently down‐regulated in malignant gliomas and thereby may support overexpression of important glioma growth‐promoting genes.
Summary Primary brain tumours are heterogeneous in histology, genetics, and outcome. Although WHO's classification of tumours of the CNS has greatly helped to standardise diagnostic criteria ...worldwide, it does not consider the substantial progress that has been made in the molecular classification of many brain tumours. Recent practice-changing clinical trials have defined a role for routine assessment of MGMT promoter methylation in glioblastomas in elderly people, and 1p and 19q codeletions in anaplastic oligodendroglial tumours. Moreover, large-scale molecular profiling approaches have identified new mutations in gliomas, affecting IDH1, IDH2, H3F3, ATRX, and CIC , which has allowed subclassification of gliomas into distinct molecular subgroups with characteristic features of age, localisation, and outcome. However, these molecular approaches cannot yet predict patients' benefit from therapeutic interventions. Similarly, transcriptome-based classification of medulloblastoma has delineated four variants that might now be candidate diseases in which to explore novel targeted agents.
Abstract
Background
Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor ...receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern.
Methods
Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas.
Results
EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations.
Conclusions
Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas.
Cerebral gliomas of World Health Organization (WHO) grade II and III represent a major challenge in terms of histological classification and clinical management. Here, we asked whether large-scale ...genomic and transcriptomic profiling improves the definition of prognostically distinct entities. We performed microarray-based genome- and transcriptome-wide analyses of primary tumor samples from a prospective German Glioma Network cohort of 137 patients with cerebral gliomas, including 61 WHO grade II and 76 WHO grade III tumors. Integrative bioinformatic analyses were employed to define molecular subgroups, which were then related to histology, molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (
IDH1/2
) mutation, 1p/19q co-deletion and telomerase reverse transcriptase (
TERT
) promoter mutations, and patient outcome. Genomic profiling identified five distinct glioma groups, including three
IDH1/2
mutant and two
IDH1/2
wild-type groups. Expression profiling revealed evidence for eight transcriptionally different groups (five
IDH1/2
mutant, three
IDH1/2
wild type), which were only partially linked to the genomic groups. Correlation of DNA-based molecular stratification with clinical outcome allowed to define three major prognostic groups with characteristic genomic aberrations. The best prognosis was found in patients with
IDH1/2
mutant and 1p/19q co-deleted tumors. Patients with
IDH1/2
wild-type gliomas and glioblastoma-like genomic alterations, including gain on chromosome arm 7q (+7q), loss on chromosome arm 10q (−10q),
TERT
promoter mutation and oncogene amplification, displayed the worst outcome. Intermediate survival was seen in patients with
IDH1/2
mutant, but 1p/19q intact, mostly astrocytic gliomas, and in patients with
IDH1/2
wild-type gliomas lacking the +7q/−10q genotype and
TERT
promoter mutation. This molecular subgrouping stratified patients into prognostically distinct groups better than histological classification. Addition of gene expression data to this genomic classifier did not further improve prognostic stratification. In summary, DNA-based molecular profiling of WHO grade II and III gliomas distinguishes biologically distinct tumor groups and provides prognostically relevant information beyond histological classification as well as
IDH1/2
mutation and 1p/19q co-deletion status.
The standard of care for anaplastic gliomas is surgery followed by radiotherapy. The NOA-04 phase III trial compared efficacy and safety of radiotherapy followed by chemotherapy at progression with ...the reverse sequence in patients with newly diagnosed anaplastic gliomas.
Patients (N = 318) were randomly assigned 2:1:1 (A:B1:B2) to receive conventional radiotherapy (arm A); procarbazine, lomustine (CCNU), and vincristine (PCV; arm B1); or temozolomide (arm B2) at diagnosis. At occurrence of unacceptable toxicity or disease progression, patients in arm A were treated with PCV or temozolomide (1:1 random assignment), whereas patients in arms B1 or B2 received radiotherapy. The primary end point was time to treatment failure (TTF), defined as progression after radiotherapy and one chemotherapy in either sequence.
Patient characteristics in the intention-to-treat population (n = 274) were balanced between arms. All histologic diagnoses were centrally confirmed. Median TTF (hazard ratio HR = 1.2; 95% CI, 0.8 to 1.8), progression-free survival (PFS; HR = 1.0; 95% CI, 0.7 to 1.3, and overall survival (HR = 1.2; 95% CI, 0.8 to 1.9) were similar for arms A and B1/B2. Extent of resection was an important prognosticator. Anaplastic oligodendrogliomas and oligoastrocytomas share the same, better prognosis than anaplastic astrocytomas. Hypermethylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) promoter (HR = 0.59; 95% CI, 0.36 to 1.0), mutations of the isocitrate dehydrogenase (IDH1) gene (HR = 0.48; 95% CI, 0.29 to 0.77), and oligodendroglial histology (HR = 0.33; 95% CI, 0.2 to 0.55) reduced the risk of progression. Hypermethylation of the MGMT promoter was associated with prolonged PFS in the chemotherapy and radiotherapy arm.
Initial radiotherapy or chemotherapy achieved comparable results in patients with anaplastic gliomas. IDH1 mutations are a novel positive prognostic factor in anaplastic gliomas, with a favorable impact stronger than that of 1p/19q codeletion or MGMT promoter methylation.
The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the ...(epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.
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•Highly divergent posttranscriptional pathway regulation in MB subgroups•Phosphoproteomic profiles reveal specific kinase activity in MB subgroups•Identification of aberrant ERBB4-SRC signaling as a hallmark of group 4 MBs•Over expression of activated SRC in the developing cerebellum induces MB
Using proteomic analyses, Forget et al. unravel divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas (MB) and identify aberrant ERBB4-SRC signaling in group 4. Expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 MB.
EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has ...been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document coexpression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.
•Rare cells within human glioblastomas express both EGFR and tumor-derived EGFRvIII•Coexpression of EGFR and EGFRvIII shows enhanced transformation in vitro and in vivo•EGFR promotes unidirectional activation of EGFRvIII, converging to activate STAT3•Coexpression of EGFR and EGFRvIII leads to a nuclear complex between vIII and STAT3
J. Neurochem. (2012) 122, 444–455.
Temozolomide (TMZ) is an alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma. Clinical benefit is more prominent in patients ...with methylation of the O6‐methyl‐guanine DNA methyltransferase (MGMT) promoter. However, all patients eventually suffer from tumor progression because their tumors become resistant to TMZ. Here, we modeled acquired TMZ resistance in glioma cells in vitro to identify underlying molecular mechanisms. To this end, the glioma cell lines LNT‐229, LN‐308, and LN‐18 were exposed repetitively to increasing concentrations of TMZ to induce a stable resistant phenotype (R) defined by clonogenic survival assays. The molecular mechanisms mediating acquired resistance were assessed by immunoblot, PCR, and flow cytometry. Rescue experiments were performed with siRNA‐mediated candidate gene silencing. We found in LN‐18 cells constitutively expressing MGMT a strong up‐regulation of MGMT levels in TMZ‐resistant cells. TMZ resistance in the MGMT‐negative cell lines LNT‐229 and LN‐308 was not associated with de novo expression of MGMT. Instead, we found a down‐regulation of several DNA mismatch‐repair proteins in resistant LNT‐229 cells. A TMZ‐resistant phenotype was also achieved by silencing selected DNA mismatch repair proteins in parental LNT‐229 cells. No obvious mechanism of resistance was identified in the third cell line, LN‐308, except for reduced methylation of LINE‐1 repetitive elements. In conclusion, we demonstrate that different molecular mechanisms may contribute to the development of acquired TMZ resistance in glioma cells, indicating the need to develop distinct strategies to overcome resistance.
Identification of molecular mechanisms leading to temozolomide resistanceAcquired resistance to temozolomide is an important challenge in the setting of therapy failure in glioblastoma patients. Here, we identify distinct mechanisms by which glioma cells become resistant to the anti‐tumor effects of temozolomide. These findings highlight the importance of different therapy‐induced molecular changes, which might be exploited for novel therapeutic approaches in the future.
Somatic mutations in the
IDH1
gene encoding cytosolic NADP+-dependent isocitrate dehydrogenase have been shown in the majority of astrocytomas, oligodendrogliomas and oligoastrocytomas of WHO grades ...II and III.
IDH2
encoding mitochondrial NADP+-dependent isocitrate dehydrogenase is also mutated in these tumors, albeit at much lower frequencies. Preliminary data suggest an importance of
IDH1
mutation for prognosis showing that patients with anaplastic astrocytomas, oligodendrogliomas and oligoastrocytomas harboring
IDH1
mutations seem to fare much better than patients without this mutation in their tumors. To determine mutation types and their frequencies, we examined 1,010 diffuse gliomas. We detected 716
IDH1
mutations and 31
IDH2
mutations. We found 165
IDH1
(72.7%) and 2
IDH2
mutations (0.9%) in 227 diffuse astrocytomas WHO grade II, 146
IDH1
(64.0%) and 2
IDH2
mutations (0.9%) in 228 anaplastic astrocytomas WHO grade III, 105
IDH1
(82.0%) and 6
IDH2
mutations (4.7%) in 128 oligodendrogliomas WHO grade II, 121
IDH1
(69.5%) and 9
IDH2
mutations (5.2%) in 174 anaplastic oligodendrogliomas WHO grade III, 62
IDH1
(81.6%) and 1
IDH2
mutations (1.3%) in 76 oligoastrocytomas WHO grade II and 117
IDH1
(66.1%) and 11
IDH2
mutations (6.2%) in 177 anaplastic oligoastrocytomas WHO grade III. We report on an inverse association of
IDH1
and
IDH2
mutations in these gliomas and a non-random distribution of the mutation types within the tumor entities.
IDH1
mutations of the R132C type are strongly associated with astrocytoma, while
IDH2
mutations predominantly occur in oligodendroglial tumors. In addition, patients with anaplastic glioma harboring
IDH1
mutations were on average 6 years younger than those without these alterations.
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