Approximately 40% of all glioblastomas have amplified the
gene, and about half of these tumors express the EGFRvIII variant. The prognostic role of EGFRvIII in
-amplified glioblastoma patients and ...changes in EGFRvIII expression in recurrent versus primary glioblastomas remain controversial, but such data are highly relevant for EGFRvIII-targeted therapies.
-amplified glioblastomas from 106 patients were assessed for EGFRvIII positivity. Changes in
amplification and EGFRvIII status from primary to recurrent glioblastomas were evaluated in 40 patients with
-amplified tumors and 33 patients with
-nonamplified tumors.
single-nucleotide variants (SNV) were assessed in 27 patients. Data were correlated with outcome and validated in 150 glioblastoma patients from The Cancer Genome Atlas (TCGA) consortium.
Sixty of 106
-amplified glioblastomas were EGFRvIII-positive (56.6%). EGFRvIII positivity was not associated with different progression-free or overall survival. EGFRvIII status was unchanged at recurrence in 35 of 40 patients with
-amplified primary tumors (87.5%). Four patients lost and one patient gained EGFRvIII positivity at recurrence. None of 33
nonamplified glioblastomas acquired
amplification or EGFRvIII at recurrence.
SNVs were frequent in
-amplified tumors, but were not linked to survival.
EGFRvIII and
SNVs are not prognostic in
-amplified glioblastoma patients.
amplification is retained in recurrent glioblastomas. Most EGFRvIII-positive glioblastomas maintain EGFRvIII positivity at recurrence. However, EGFRvIII expression may change in a subset of patients at recurrence, thus repeated biopsy with reassessment of EGFRvIII status is recommended for patients with recurrent glioblastoma to receive EGFRvIII-targeting agents.
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Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, ...characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma‐tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS‐based mutation detection was optimized for application on formalin‐fixed paraffin‐embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α‐thalassemia/mental retardation syndrome X‐linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We studied how intratumoral genetic heterogeneity shapes tumor growth and therapy response for isocitrate dehydrogenase (IDH)-wild-type glioblastoma, a rapidly regrowing tumor. We inferred the ...evolutionary trajectories of matched pairs of primary and relapsed tumors based on deep whole-genome-sequencing data. This analysis suggests both a distant origin of de novo glioblastoma, up to 7 years before diagnosis, and a common path of early tumorigenesis, with one or more of chromosome 7 gain, 9p loss, or 10 loss, at tumor initiation. TERT promoter mutations often occurred later as a prerequisite for rapid growth. In contrast to this common early path, relapsed tumors acquired no stereotypical pattern of mutations and typically regrew from oligoclonal origins, suggesting sparse selective pressure by therapeutic measures.
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•We inferred evolutionary trajectories of pairs of primary/relapsed glioblastomas•Chromosome 7 gain, 9p loss, or 10 loss commonly occurred at tumor initiation•TERT promoter mutations often occurred later as a prerequisite for rapid growth•Relapsed tumors typically regrew from oligoclonal origins
By analyzing 21 paired primary and locally relapsed IDH-wild-type glioblastomas (GBM), Körber et al. show that most GBM initiate by gains and losses of specific chromosomes; TERT promoter mutations often occur later as a prerequisite for rapid growth, and relapsed GBM acquire few stereotypical mutations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Molecular genetic aberrations in the phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, ...novel therapeutic approaches targeting this pathway in glioblastoma have not been successful. We hypothesized that molecular profiling in combination with in vitro drug sensitivity testing allows to identify signatures associated with sensitivity or resistance to PI3K/mTOR pathway inhibition. We analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition and proliferation, clonogenicity, or spherogenicity as readouts, in human long‐term glioma cell (LTC) lines and glioma‐initiating cells (GIC). Cultured glioma cells were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan‐PI3K/mTOR antagonist. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E‐binding protein 1 (p4E‐BP1), and high levels of Ser9‐phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3β) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition. In vivo studies confirmed the anti‐glioma activity of PQR309 alone or in combination with AKT inhibition in the orthotopic LN‐229 glioma xenograft model in nude mice. These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down‐regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.
Molecular genetic aberrations in the PI3K/AKT/mTOR pathway are common in glioblastoma. We hypothesized that molecular profiling in combination with in vitro drug sensitivity testing may allow to identify signatures associated with sensitivity or resistance to PI3K/mTOR pathway inhibition. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor 4E‐BP1, and high levels of Ser9‐phosphorylated (inactive) GSK3β were more sensitive to PQR309, a dual pan‐PI3K/mTOR antagonist in vitro.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Classification of gliomas involves the combination of histological features with molecular biomarkers to establish an integrated histomolecular diagnosis. Here, we report on the application and ...validation of a set of molecular assays for glioma diagnostics based on digital PCR technology using the QX200™ Droplet Digital™ PCR (ddPCR) system. The investigated ddPCR-based assays enable the detection of diagnostically relevant glioma-associated mutations in the IDH1, IDH2, H3-3A, BRAF, and PRKCA genes, as well as in the TERT promoter. In addition, ddPCR-based assays assessing diagnostically relevant copy number alterations were studied, including 1p/19q codeletion, gain of chromosome 7 and loss of chromosome 10 (+ 7/-10), EGFR amplification, duplication of the BRAF locus, and CDKN2A homozygous deletion. Results obtained by ddPCR were validated by other methods, including immunohistochemistry, Sanger sequencing, pyrosequencing, microsatellite analyses for loss of heterozygosity, as well as real-time PCR- or microarray-based copy number assays. Particular strengths of the ddPCR approach are (1) its high analytical sensitivity allowing for reliable detection of mutations even with low mutant allele frequencies, (2) its quantitative determination of mutant allele frequencies and copy number changes, and (3) its rapid generation of results within a single day. Thus, in line with other recent studies our findings support ddPCR analysis as a valuable approach for molecular glioma diagnostics in a fast, quantitative and highly sensitive manner.
This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. Patients from the HIT2000 cooperative clinical trial ...were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (
n
= 127), and a test (
n
= 57) dataset in order to build and test a risk score for this population. Independent validation was performed in a non-overlapping cohort (
n
= 83). All samples were subjected to thorough histopathological investigation,
CTNNB1
mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status,
MYC
amplification, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified
CTNNB1
sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (
CTNNB1
mutation, extensive nodularity) or unfavorable (
MYC
amplification) markers, a risk score for the remaining “intermediate molecular risk” population dependent on age, M-stage, pattern of synaptophysin expression, and
MYCN
copy-number status was identified, with speckled synaptophysin expression indicating worse outcome. Test and independent validation of the score confirmed significant discrimination of patients by risk profile. Methylation subgrouping and
CTNNB1
mutation status represent robust tools for the risk stratification of medulloblastoma. A simple clinico-pathological risk score was identified, which was confirmed in a test set and by independent clinical validation.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The epidermal growth factor receptor vIII mutant (EGFRvIII) is found in ∼50% of all EGFR‐amplified glioblastomas and constitutes a tumor‐specific therapeutic target. To assess molecular testing ...approaches and the prognostic role of EGFRvIII in patients treated according to current standards of care, we compared different EGFRvIII detection methods and correlated EGFRvIII status with outcome in a prospective patient cohort of the German Glioma Network. In total, 184 newly diagnosed glioblastoma patients were investigated for EGFR amplification and for expression of EGFR and EGFRvIII by immunohistochemistry. Further, the EGFRvIII status was additionally studied by multiplex ligation‐dependent probe amplification (MLPA) analysis and reverse transcription‐PCR (RT‐PCR). Immunohistochemistry demonstrated EGFRvIII in 34 of 184 patients (18%). RT‐PCR or MLPA analysis detected four additional EGFRvIII‐positive patients. Overall, RT‐PCR and immunohistochemistry were more sensitive for EGFRvIII detection than MLPA. EGFRvIII status was not associated with progression‐free and overall survival. EGFRvIII also had no prognostic significance in the subgroup of patients who were free from progression after concomitant radiochemotherapy and thus would be eligible for the ongoing ACT IV EGFRvIII vaccination trial. Age, extent of resection and O6‐methylguanine DNA methyltransferase (MGMT) promoter methylation status appeared to be less prognostic in EGFRvIII‐positive patients. Thus, EGFRvIII positivity is not a major negative prognostic factor in glioblastoma patients treated according to current standards of care. Data from phase II EGFRvIII‐targeted vaccination trials compare favorably with the present contemporary results, supporting the further exploration of EGVRvIII vaccination in newly diagnosed glioblastoma.
What's new?
The epidermal growth factor receptor deletion mutant vIII (EGFRvIII) is expressed in 20–25% of primary glioblastomas and constitutes a promising tumor‐specific target for immunotherapy. Here, the authors characterize immunohistochemistry and reverse transcription‐PCR as the most sensitive methods for diagnostic EGFRvIII detection. They also show that EGFRvIII positivity is not associated with survival in glioblastoma patients treated according to current standards of care. Data from phase II EGFRvIII‐targeted vaccination trials compare favorably with the presented results, supporting the further exploration of EGVRvIII vaccination in newly diagnosed glioblastoma.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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