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, 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
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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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, SBCE, SBMB, UL, UM, UPUK
O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma. We investigated whether this marker furthermore defines a molecularly distinct ...tumor subtype with clinically different outcome.
We analyzed copy number variation (CNV) and methylation profiles of 1095 primary and 92 progressive isocitrate dehydrogenase wildtype glioblastomas, including paired samples from 49 patients. DNA mutation data from 182 glioblastoma samples of The Cancer Genome Atlas (TCGA) and RNA expression from 107 TCGA and 55 Chinese Glioma Genome Atlas samples were analyzed.
Among untreated glioblastomas, MGMT promoter methylated (mMGMT) and unmethylated (uMGMT) tumors did not show different CNV or specific gene mutations, but a higher mutation count in mMGMT tumors. We identified 3 methylation clusters. Cluster 1 showed the highest average methylation and was enriched for mMGMT tumors. Seventeen genes including gastrulation brain homeobox 2 (GBX2) were found to be hypermethylated and downregulated on the mRNA level in mMGMT tumors. In progressive glioblastomas, platelet derived growth factor receptor alpha (PDGFRA) and GLI2 amplifications were enriched in mMGMT tumors. Methylated MGMT tumors gain PDGFRA amplification of PDGFRA, whereas uMGMT tumors with amplified PDGFRA frequently lose this amplification upon progression. Glioblastoma patients surviving <6 months and with mMGMT harbored less frequent epidermal growth factor receptor (EGFR) amplifications, more frequent TP53 mutations, and a higher tumor necrosis factor-nuclear factor-kappaB (TNF-NFκB) pathway activation compared with patients surviving >12 months.
MGMT promoter methylation status does not define a molecularly distinct glioblastoma subpopulation among untreated tumors. Progressive mMGMT glioblastomas and mMGMT tumors of patients with short survival tend to have more unfavorable molecular profiles.
Abstract
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 had limited success to date. Here we analyzed the molecular mechanisms determining sensitivity to PI3K/AKT/mTOR inhibition using gene silencing or pharmacological inhibition by assessing target inhibition, modulation of down-stream signaling pathways, viability, proliferation, and clonogenicity (sphere formation) in human isocitrate dehydrogenase (IDH) wild-type long-term cell (LTC) lines and glioma-initiating cells (GIC). Glioma cells including GIC in particular were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/AKT/mTOR antagonist in vitro. 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, PQR309 acted synergistically in combination with AKT inhibitors to inhibit clonogenicity or spherogenicity in vitro, indicating that persistent AKT activity may represent an escape mechanism from PI3K/AKT/mTOR-targeted therapy. In vivo studies confirmed the anti-glioma activity of PQR309 alone or in combination with AKT inhibition in the orthotopic LN-229 glioma model. Altogether, these data may help to stratify or enrich for patients likely to benefit from PI3K/AKT/mTOR inhibition in future clinical trials of targeted therapy in glioblastoma.
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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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The Antarctic Peninsula experiences a fast retreat of glaciers, which correlates with an increased release of particles and related increased sedimentation and, thus, a decrease in the available ...photosynthetic active radiation (PAR, 400–700 nm) for benthic primary production. We investigated how changes in the general sedimentation and shading patterns affect the primary production by benthic microalgae, the microphytobenthos. In order to determine potential net primary production and respiration of the microphytobenthic community, sediment cores from locations exposed to different sedimentation rates and shading were exposed to PAR of 0–70 µmol photons m-2 s-1. Total oxygen fluxes and microphytobenthic diatom community structure, density, and biomass were determined. Our study revealed that the net primary production of the microphytobenthos decreased with increasing sedimentation and shading, while the microphytobenthic diatom density and composition remained similar. By comparing our experimental results with in situ measured PAR intensities, we furthermore assessed the microphytobenthic primary production as an important carbon source within Potter Cove’s benthic ecosystem. We propose that the microphytobenthic contribution to the total primary production may drop drastically due to Antarctic glacial retreat and correlated sedimentation and shading, with yet unknown consequences for the benthic heterotrophic community, its structure, and diversity.
The spread of multidrug resistant organisms (MDRO) is a global healthcare challenge. Nosocomial outbreaks caused by MDRO are an important contributor to this threat. Computer-based applications ...facilitating outbreak detection can be essential to address this issue. To allow application reusability across institutions, the various heterogeneous microbiology data representations needs to be transformed into standardised, unambiguous data models. In this work, we present a multi-centric standardisation approach by using openEHR as modelling standard. Data models have been consented in a multicentre and international approach. Participating sites integrated microbiology reports from primary source systems into an openEHR-based data platform. For evaluation, we implemented a prototypical application, compared the transformed data with original reports and conducted automated data quality checks. We were able to develop standardised and interoperable microbiology data models. The publicly available data models can be used across institutions to transform real-life microbiology reports into standardised representations. The implementation of a proof-of-principle and quality control application demonstrated that the new formats as well as the integration processes are feasible. Holistic transformation of microbiological data into standardised openEHR based formats is feasible in a real-life multicentre setting and lays the foundation for developing cross-institutional, automated outbreak detection systems.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Ultraviolet radiation (UVR) research on marine macroalgae has hithero focussed on physiological effects at the organism level, while little is known on the impact of UV radiation on macroalgal ...assemblages and even less on interactive effects with other community drivers, e.g. consumers. Field experiments on macrobenthos are scarce, particularly in the Antarctic region. Therefore, the effects of UVR and consumers (mainly limpets were excluded) on early successional stages of a hard bottom macroalgal community on King George Island, Antarctica, were studied. In a two‐factorial design experimental units (1) ambient radiation, 280–700 nm; (2) ambient minus UVB, 320–700 nm and (3) ambient minus UVR, 400–700 nm vs. consumer–no consumer were installed between November 2004 and March 2005 (n= 4 plus controls). Dry mass, species richness, diversity and composition of macroalgal assemblages developing on ceramic tiles were followed. Consumers significantly suppressed green algal recruits and total algal biomass but increased macroalgal richness and diversity. Both UVA and UVB radiation negatively affected macroalgal succession. UVR decreased the density of Monostroma hariotii germlings in the first 10 weeks of the experiment, whereas the density of red algal recruits was significantly depressed by UVR at the end of the study. After 106 days macroalgal diversity was significantly higher in UV depleted than in UV‐exposed assemblages. Furthermore, species richness was significantly lower in the UV treatments and species composition differed significantly between the UV‐depleted and the UV‐exposed treatment. Marine macroalgae are very important primary producers in coastal ecosystems, serving as food for herbivores and as habitat for many organisms. Both, UVR and consumers significantly shape macroalgal succession in the Antarctic intertidal. Consumers, particularly limpets can mediate negative effects of ambient UVR on richness and diversity till a certain level. UVB radiation in general and an increase of this short wavelength due to stratospheric ozone depletion in particular may have the potential to affect the zonation, composition and diversity of Antarctic intertidal seaweeds altering trophic interactions in this system.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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