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Since 2013 next-generation sequencing (NGS) targeting genes mutated in diffuse gliomas is part of routine diagnostics in our institute. In the present report, we evaluate the use of this custom tailored NGS platform on 434 samples. The NGS panel assesses mutations in ATRX, CIC, EGFR, FUBP1, NOTCH1, PTEN; H3F3A, IDH1/2, PIK3CA, and BRAF, amplifications in EGFR or MDM2 and copy number alterations (CNA) of chromosome 1p, 7, 10 and 19q. TERT promoter mutations were assessed separately when indicated. Of the 433 samples of individual tumors with NGS data available, 176 cases were diagnosed as grade 2 or 3 glioma (40.6) and in 201 patients a glioblastoma (46.4%). Of the remaining 56 patients, 22 had inconclusive histology. In 378 cases (87.1%) a diagnosis solely based on glioma-targeted NGS could be established and resulted in a different diagnosis in ~ 1/4 of the cases. In 17 out of 22 cases without a conclusive histological diagnosis NGS resulted in a molecular diagnosis.The current study on a large cohort of patients confirms the diagnostic strength of the platform we developed, with a clear separation of glioma subgroups with different outcomes. It demonstrates the diagnostic value and the efficiency of glioma-targeted NGS for routine glioma diagnostics allowing with a single assay a glioma diagnosis in the large majority of cases. It allows in one run the molecular assessments required for the WHO classification of diffuse gliomas, including the recent recommendations to assess copy number alterations of chromosome 7 and 10, and of the TERT promoter region in IDHwt lower grade glioma.

Background: At current prognostication of low grade glioma remains suboptimal and might be improved with additional markers. These may guide treatment decisions, in particular on early adjuvant therapy versus wait and see after surgery., Methods: We used a targeted Next-Generation Sequencing panel to assess mutational and copy number status of selected genes and chromosomes in a consecutive series of adult grade II supratentorial glioma, and assessed the impact of molecular markers of interest on overall survival., Results: 207 IDH mutated grade II glioma samples were analyzed with a median follow-up of 6.9 years. Loss of region 9p21.3 did not show a correlation with outcome in IDH mutated 1p/19q-codeleted oligodendroglioma or IDH mutated astrocytoma. We found a significant shorter overall survival with univariable analysis in IDH mutated astrocytoma patients with trisomy of chromosome 7 (Log rank P = 0.044) and in IDH mutated 1p/19q-codeleted oligodendroglioma patients with a PTEN mutation (Log rank P = 0.033). We could not validate these findings in multivariate analysis or in the TCGA dataset., Conclusions: Loss of 9p21.3 is not associated with outcome in a molecularly defined cohort of grade II glioma and therefore it remains unclear if loss of 9p21.3 can be used as additional marker of anaplasia or to guide treatment decisions. Trisomy of chromosome 7 in IDH mutated astrocytoma and PTEN mutations in IDH mutated oligodendroglioma are potential markers of poor prognosis, but require confirmation in larger series.

Background: Extensive resections in low-grade glioma (LGG) are associated with improved overall survival (OS). However, World Health Organization (WHO) classification of gliomas has been completely revised and is now predominantly based on molecular criteria. This requires reevaluation of the impact of surgery in molecularly defined LGG subtypes., Methods: We included 228 adults who underwent surgery since 2003 for a supratentorial LGG. Pre- and postoperative tumor volumes were assessed with semiautomatic software on T2-weighted images. Targeted next-generation sequencing was used to classify samples according to current WHO classification. Impact of postoperative volume on OS, corrected for molecular profile, was assessed using a Cox proportional hazards model., Results: Median follow-up was 5.79 years. In 39 (17.1%) histopathologically classified gliomas, the subtype was revised after molecular analysis. Complete resection was achieved in 35 patients (15.4%), and in 54 patients (23.7%) only small residue (0.1-5.0 cm3) remained. In multivariable analysis, postoperative volume was associated with OS, with a hazard ratio of 1.01 (95% CI: 1.002-1.02; P = 0.016) per cm3 increase in volume. The impact of postoperative volume was particularly strong in isocitrate dehydrogenase (IDH) mutated astrocytoma patients, where even very small postoperative volumes (0.1-5.0 cm) already negatively affected OS., Conclusion: Our data provide the necessary reevaluation of the impact of surgery in molecularly defined LGG and support maximal resection as first-line treatment for molecularly defined LGG. Importantly, in IDH mutated astrocytoma, even small postoperative volumes have negative impact on OS, which argues for a second-look operation in this subtype to remove minor residues if safely possible., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Cancer cells are genomically unstable and accumulate tumor type-specific molecular aberrations, which may represent hallmarks for predicting prognosis and targets for therapy. Co-deletion of chromosomes 1p and 19q marks gliomas with an oligodendroglioma component and predicts a better prognosis and response to chemotherapy. In the current study, we present a novel method to detect chromosome 1p/19q co-deletion or loss of heterozygosity (LOH) in a diagnostic setting, based on single-nucleotide polymorphism (SNP) analysis and next-generation sequencing (NGS). We selected highly polymorphic SNPs distributed evenly over both chromosome arms. To experimentally determine the sensitivity and specificity of targeted SNP analysis, we used DNAs extracted from 49 routine formalin-fixed, paraffin-embedded glioma tissues and compared the outcome with diagnostic microsatellite-based LOH analysis and calculated estimates. We show that targeted SNP analysis by NGS allows reliable detection of 1p and/or 19q deletion in a background of 70% of normal cells according to calculated outcomes, is more sensitive than microsatellite-based LOH analysis, and requires much less DNA. This specific and sensitive SNP assay is broadly applicable for simultaneous allelic imbalance analysis of multiple genomic regions and can be incorporated easily into NGS mutation analyses. The combined mutation and chromosomal imbalance analysis in a single NGS assay is suited perfectly for routine glioma diagnostics and other diagnostic molecular pathology applications., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Histopathological diagnosis of diffuse gliomas is subject to interobserver variation and correlates modestly with major prognostic and predictive molecular abnormalities. We investigated a series of patients with locally diagnosed anaplastic oligodendroglial tumors included in the EORTC phase III trial 26951 on procarbazine/lomustine/vincristine (PCV) chemotherapy to explore the diagnostic, prognostic, and predictive value of targeted next-generation sequencing (NGS) in diffuse glioma and to assess the prognostic impact of FUBP1 and CIC mutations., Methods: Mostly formalin-fixed paraffin-embedded samples were tested with targeted NGS for mutations in ATRX, TP53, IDH1, IDH2, CIC, FUBP1, PI3KC, TERT, EGFR, H3F3A, BRAF, PTEN, and NOTCH and for copy number alterations of chromosomes 1p, 19q, 10q, and 7. TERT mutations were also assessed, with PCR., Results: Material was available from 139 cases, in 6 of which results were uninformative. One hundred twenty-six tumors could be classified: 20 as type II (IDH mutation [mut], "astrocytoma"), 49 as type I (1p/19q codeletion, "oligodendroglioma"), 55 as type III (7+/10q- or TERTmut and 1p/19q intact, "glioblastoma"), and 2 as childhood glioblastoma (H3F3Amut), leaving 7 unclassified (total 91% classified). Molecular classification was of clear prognostic significance and correlated better with outcome than did classical histopathology. In 1p/19q codeleted tumors, outcome was not affected by CIC and FUBP1 mutations. MGMT promoter methylation remained the most predictive factor for survival benefit of PCV chemotherapy., Conclusion: Targeted NGS allows a clinically relevant classification of diffuse glioma into groups with very different outcomes. The diagnosis of diffuse glioma should be primarily based on a molecular classification, with the histopathological grade added to it. Future discussion should primarily aim at establishing the minimum requirements for molecular classification of diffuse glioma., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)