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The mainstay of treatment for adult patients with gliomas, glioneuronal and neuronal tumors consists of combinations of surgery, radiotherapy, and chemotherapy. For many systemic cancers, targeted treatments are a part of the standard of care, however, the predictive significance of most of these targets in central nervous system (CNS) tumors remains less well-studied. Despite that, there is increasing use of advanced molecular diagnostics that identify potential targets, and tumor-agnostic regulatory approvals on targets also present in CNS tumors have been granted. This raises the question of when and for which targets it is meaningful to test in adult patients with CNS tumors. This evidence-based guideline reviews the evidence available for targeted treatment for alterations in the RAS/MAPK pathway (BRAF, NF1), in growth factor receptors (EGFR, ALK, fibroblast growth factor receptor (FGFR), neurotrophic tyrosine receptor kinase (NTRK), platelet-derived growth factor receptor alpha, and ROS1), in cell cycle signaling (CDK4/6, MDM2/4, and TSC1/2) and altered genomic stability (mismatch repair, POLE, high tumor mutational burden (TMB), homologous recombination deficiency) in adult patients with gliomas, glioneuronal and neuronal tumors. At present, targeted treatment for BRAF p.V600E alterations is to be considered part of the standard of care for patients with recurrent gliomas, pending regulatory approval. For approved tumor agnostic treatments for NTRK fusions and high TMB, the evidence for efficacy in adult patients with CNS tumors is very limited, and treatment should preferably be given within prospective clinical registries and trials. For targeted treatment of CNS tumors with FGFR fusions or mutations, clinical trials are ongoing to confirm modest activity so far observed in basket trials. For all other reviewed targets, evidence of benefit in CNS tumors is currently lacking, and testing/treatment should be in the context of available clinical trials.

BackgroundGliomas with IDH1/2 mutations without 1p19q codeletion have been identified as the distinct diagnostic entity of IDH mutant astrocytoma (IDHmut astrocytoma). Homozygous deletion of Cyclin-dependent kinase 4 inhibitor A/B (CDKN2A/B) has recently been incorporated in the grading of these tumors. The question of whether histologic parameters still contribute to prognostic information on top of the molecular classification, remains unanswered. Here we evaluated consensus histologic parameters for providing additional prognostic value in IDHmut astrocytomas.MethodsAn international panel of seven neuropathologists scored 13 well-defined histologic features in virtual microscopy images of 192 IDHmut astrocytomas from EORTC trial 22033-26033 (low-grade gliomas) and 263 from EORTC 26053 (CATNON) (1p19q non-codeleted anaplastic glioma). For 192 gliomas the CDKN2A/B status was known. Consensus (agreement ≥ 4/7 panelists) histologic features were tested together with homozygous deletion (HD) of CDKN2A/B for independent prognostic power.ResultsAmong consensus histologic parameters, the mitotic count (cut-off of 2 mitoses per 10 high power fields standardized to a field diameter of 0.55 mm and an area of 0.24 mm2) significantly influences PFS (P = .0098) and marginally the OS (P = .07). Mitotic count also significantly affects the PFS of tumors with HD CDKN2A/B, but not the OS, possibly due to limited follow-up data.ConclusionThe mitotic index (cut-off 2 per 10 40× HPF) is of prognostic significance in IDHmut astrocytomas without HD CDKN2A/B. Therefore, the mitotic index may direct the therapeutic approach for patients with IDHmut astrocytomas with native CDKN2A/B status.

Background Survival in patients with IDH1/2-mutant (mt) anaplastic astrocytomas is highly variable. We have used the prospective phase 3 CATNON trial to identify molecular factors related to outcome in IDH1/2mt anaplastic astrocytoma patients. Methods The CATNON trial randomized 751 adult patients with newly diagnosed 1p/19q non-codeleted anaplastic glioma to 59.4 Gy radiotherapy +/− concurrent and/or adjuvant temozolomide. The presence of necrosis and/or microvascular proliferation was scored at central pathology review. Infinium MethylationEPIC BeadChip arrays were used for genome-wide DNA methylation analysis and the determination of copy number variations (CNV). Two DNA methylation-based tumor classifiers were used for risk stratification. Next-generation sequencing (NGS) was performed using 1 of the 2 glioma-tailored NGS panels. The primary endpoint was overall survival measured from the date of randomization. Results Full analysis (genome-wide DNA methylation and NGS) was successfully performed on 654 tumors. Of these, 432 tumors were IDH1/2mt anaplastic astrocytomas. Both epigenetic classifiers identified poor prognosis patients that partially overlapped. A predictive prognostic Cox proportional hazard model identified that independent prognostic factors for IDH1/2mt anaplastic astrocytoma patients included; age, mini-mental state examination score, treatment with concurrent and/or adjuvant temozolomide, the epigenetic classifiers, PDGFRA amplification, CDKN2A/B homozygous deletion, PI3K mutations, and total CNV load. Independent recursive partitioning analysis highlights the importance of these factors for patient prognostication. Conclusion Both clinical and molecular factors identify IDH1/2mt anaplastic astrocytoma patients with worse outcome. These results will further refine the current WHO criteria for glioma classification.

Background IDH1/2 wildtype (IDHwt) glioma WHO grade 2 and 3 patients with pTERT mutation and/or EGFR amplification and/or + 7/−10 chromosome gain/loss have a similar overall survival time as IDHwt glioblastoma patients, and are both considered glioblastoma IDHwt according to the WHO 2021 classification. However, differences in seizure onset have been observed. This study aimed to compare the course of epilepsy in the 2 glioblastoma subtypes. Methods We analyzed epilepsy data of an existing cohort including IDHwt histologically lower-grade glioma WHO grade 2 and 3 with molecular glioblastoma-like profile (IDHwt hLGG) and IDHwt glioblastoma patients. Primary outcome was the incidence proportion of epilepsy during the disease course. Secondary outcomes included, among others, onset of epilepsy, number of seizure days, and antiepileptic drug (AED) polytherapy. Results Out of 254 patients, 78% (50/64) IDHwt hLGG and 68% (129/190) IDHwt glioblastoma patients developed epilepsy during the disease (P = .121). Epilepsy onset before histopathological diagnosis occurred more frequently in IDHwt hLGG compared to IDHwt glioblastoma patients (90% vs 60%, P Conclusions Although the incidence proportion of epilepsy during the entire disease course is similar, IDHwt hLGG patients show a significantly higher incidence of epilepsy before diagnosis and a significantly longer median time between first seizure and diagnosis compared to IDHwt glioblastoma patients, indicating a distinct clinical course.

Background The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) has recommended that isocitrate dehydrogenase 1 and 2 wildtype (IDH1/2wt) diffuse lower-grade gliomas (LGGs) World Health Organization (WHO) grade II or III that present with (i) a telomerase reverse transcriptase promoter mutation (pTERTmt), and/or (ii) gain of chromosome 7 combined with loss of chromosome 10, and/or (iii) epidermal growth factor receptor (EGFR) amplification should be reclassified as diffuse astrocytic glioma, IDH1/2 wildtype, with molecular features of glioblastoma, WHO grade IV (IDH1/2wt astrocytomas WHO IV). This paper describes the overall survival (OS) of IDH1/2wt astrocytoma WHO IV patients, and more in detail patients with tumors with pTERTmt only. Methods In this retrospective multicenter study, we compared the OS of 71 IDH1/2wt astrocytomas WHO IV patients, with radiological characteristics of LGGs, with the OS of 197 IDH1/2wt glioblastoma patients. Moreover, we compared the OS of 22 pTERTmt only astrocytoma patients with the OS of the IDH1/2wt glioblastoma patients. Results Median OS was similar for IDH1/2wt astrocytoma WHO IV patients (23.8 mo) and IDH1/2wt glioblastoma patients (19.2 mo) (Cox proportional hazards model: hazard ratio [HR] 1.27, 95% CI: 0.85–1.88, P = 0.242). OS was also similar in patients with IDH1/2wt astrocytomas WHO IV, pTERTmt only, and IDH1/2wt glioblastomas (HR 1.15, 95% CI: 0.64–2.10, P = 0.641). Conclusions The presented data confirm the cIMPACT-NOW recommendation and we propose that IDH1/2wt astrocytomas WHO IV in the absence of other qualifying mutations should be classified as IDH1/2wt glioblastomas.

Background Depatuxizumab mafodotin (Depatux-M) is a tumor-specific antibody–drug conjugate consisting of an antibody (ABT-806) directed against activated epidermal growth factor receptor (EGFR) and the toxin monomethylauristatin-F. We investigated Depatux-M in combination with temozolomide or as a single agent in a randomized controlled phase II trial in recurrent EGFR amplified glioblastoma. Methods Eligible were patients with centrally confirmed EGFR amplified glioblastoma at first recurrence after chemo-irradiation with temozolomide. Patients were randomized to either Depatux-M 1.25 mg/kg every 2 weeks intravenously, or this treatment combined with temozolomide 150–200 mg/m2 day 1–5 every 4 weeks, or either lomustine or temozolomide. The primary endpoint of the study was overall survival. Results Two hundred sixty patients were randomized. In the primary efficacy analysis with 199 events (median follow-up 15.0 mo), the hazard ratio (HR) for the combination arm compared with the control arm was 0.71 (95% CI = 0.50, 1.02; P = 0.062). The efficacy of Depatux-M monotherapy was comparable to that of the control arm (HR = 1.04, 95% CI = 0.73, 1.48; P = 0.83). The most frequent toxicity in Depatux-M treated patients was a reversible corneal epitheliopathy, occurring as grades 3–4 adverse events in 25–30% of patients. In the long-term follow-up analysis with median follow-up of 28.7 months, the HR for the comparison of the combination arm versus the control arm was 0.66 (95% CI = 0.48, 0.93). Conclusion This trial suggests a possible role for the use of Depatux-M in combination with temozolomide in EGFR amplified recurrent glioblastoma, especially in patients relapsing well after the end of first-line adjuvant temozolomide treatment. (NCT02343406)

Background Precision medicine trials targeting the epidermal growth factor receptor (EGFR) in glioblastoma patients require selection for EGFR-amplified tumors. However, there is currently no gold standard in determining the amplification status of EGFR or variant III (EGFRvIII) expression. Here, we aimed to determine which technique and which cutoffs are suitable to determine EGFR amplification status. Methods We compared fluorescence in-situ hybridization (FISH) and real-time quantitative (RT-q)PCR data from patients screened for trial inclusion into the Intellance 2 clinical trial, with data from a panel-based next generation sequencing (NGS) platform (both DNA and RNA). Results By using data from >1000 samples, we show that at least 50% of EGFR amplified nuclei should be present to define EGFR gene amplification by FISH. Gene amplification (as determined by FISH) correlates with EGFR expression levels (as determined by RT-qPCR) with receiver operating characteristics analysis showing an area under the curve of up to 0.902. EGFR expression as assessed by RT-qPCR therefore may function as a surrogate marker for EGFR amplification. Our NGS data show that EGFR copy numbers can strongly vary between tumors, with levels ranging from 2 to more than 100 copies per cell. Levels exceeding 5 gene copies can be used to define EGFR-amplification by NGS; below this level, FISH detects very few (if any) EGFR amplified nuclei and none of the samples express EGFRvIII. Conclusion Our data from central laboratories and diagnostic sequencing facilities, using material from patients eligible for clinical trial inclusion, help define the optimal cutoff for various techniques to determine EGFR amplification for diagnostic purposes.

Background Evidence suggests that smaller residual tumor volumes after initial surgery are associated with longer survival in patients with astrocytoma, IDH-mutant, grade II. For IDH-mutant glioma of higher grade but also for oligodendroglioma, IDH-mutant, and 1p/19q codeleted, the impact on survival of more residual tumor is unclear. This could be related to the limited follow-up of patients with oligodendroglioma that typically have long overall survival. We expanded a previously published series of patients increase the duration of follow-up and the spectrum of tumor grades. Material and Methods Single center observational cohort study of patients with adult-type diffuse glioma, IDH-mutant, all WHO-2021 grades, from 2003 to 2021 . Molecular characteristics were assessed by next-generation targeted sequencing. Tumor volumes were calculated by semi-automatic 3D segmentation on pre- and postoperative MRI-scans. Associations between prognostic factors and survival were assessed using univariate and multivariate Cox proportional hazards models. Results 384 patients with IDH-mutant glioma (174 oligodendroglioma, 212 astrocytoma) were followed for a median of 7 years. Median age at diagnosis was 41 years (IQR 33 - 55). Significant prognostic factors for survival were Karnofsky performance status, 1p19q-status and post-operative tumor volume. Smaller post-operative tumor volume was associated with better survival, both in astrocytoma and oligodendroglioma. Age, tumor grade and MRI-contrast enhancement were not associated with survival. Conclusion Lower residual tumor volume after first surgery is a strong prognostic factor in all IDH-mutant glioma subgroups, even in patients with very small residual tumor. Importantly, age and histological tumor grade were not significantly associated with survival. The data serves as further support for optimal initial resections of tumors suspected for IDH-mutant glioma, also in oligodendroglioma.

Background The fifth edition of the WHO Classification of Tumors of the Central Nervous System (WHO CNS 5), includes molecular parameters for both diagnosis and grading in addition to histological features. For IDH-mutant astrocytoma, homozygous deletion (HD) of CDKN2A/B now results in WHO grade 4, even in the absence of microvascular proliferation or necrosis. CDKN2A/B deletions can be determined by various techniques including shallow and targeted sequencing, and using genome wide DNA-methylation arrays. Various algorithms to call deletions also exist for each platform. Concordance between the various techniques and algorithms is however unknown. Because of the importance to properly call CDKN2A/B deletions, we compared two techniques to call HD in IDH-mutant astrocytoma patients. Methods Samples from 110 IDH-mutant astrocytoma patients enrolled in the GLASS-NL study, and therefore samples from at least two surgical resections per patient, were available. Overall survival (OS) was measured from date of first surgery. Both DNA-methylation data and shallow whole-genome sequencing (sWGS) was collected from 219 samples from 101 patients. For DNA-methylation analysis, HD of CDKN2A/B was defined by Results Method 1 and method 2 called an HD of CDKN2A/B in 39 and 33 cases, respectively; method 1 called an HD of CDKN2A/B in 9 cases in which method 2 did not detect an alteration. The agreement was strong with 95% agreement and κ = 0.842. Method 3 called an HD of CDKN2A/B in 45 cases of which 12 and 19 were not detected by method 1 and method 2 respectively. Method 3 showed an almost perfect level of agreement with method 1, with 97% agreement and κ = 0.911, and a moderate agreement with method 2, with 89% agreement and κ = 0.671. Survival analysis showed that there was no significant difference in survival when patients were stratified by CDKN2A/B status of the initial sample of all methods. However, when stratified by CDKN2A/B status of the first recurrence, all methods showed a stronger association with OS (method 1: p = 0.001, HR = 2.61[1.44-4.72]; method 2: p = 0.0024, HR = 2.34[1.33-4.11]; method 3: p = 0.0099, HR = 2.34[1.2-4.55]). Conclusion Different methods and different cutoffs to determine the presence of a HD in CDKN2A/B may result in different test outcomes. Quality of the DNA, tumor cell percentage of the sample and ploidy are likely to influence the call of homozygous CDKN2A/B deletion. Ring tests are recommended to improve reliability.

Background The evolutionary processes that drive tumor progression in patients with IDH-mutant astrocytoma remain largely unclear. The GLASS-NL consortium was initiated to gain insight into the molecular mechanisms underlying glioma evolution and to identify markers of progression in IDH-mutant astrocytomas. Ultimately, such markers can assist clinical decision making. Here, we present the DNA methylation profiling, RNA-sequencing and shallow whole-genome sequencing (sWGS) of samples included in the GLASS-NL study. Methods Eligible were patients with an IDH-mutant, 1p19q non-codeleted, astrocytoma at first diagnosis who underwent surgical resection of the tumor at least twice separated by >6 months, and of whom paired tumor samples were available for analyses. Overall survival (OS) was measured from date of initial surgery. DNA methylation profiling was performed with the 850kEPIC array, and transcriptome and sWGS by NGS. After quality control, DNA methylation data of 103, expression data of 91, and sWGS data of 92 patients was available for further analysis. Methylation classes were determined according to Capper et al. and copy number alterations (CNAs) were extracted from both sWGS and DNA-methylation data. Results 110 patients were identified from various medical centers in the Netherlands. The median time between surgical resections was 41.9 months (IQR:26.5-65.9) and after initial surgery, 63% and 22% of the patients were treated with radiotherapy or chemotherapy respectively. The proportion of samples assigned to the high grade methylation class increased ~three-fold at recurrence. 83% of patients that progressed from low to high grade, received treatment prior to recurrent surgery, as compared to 53% of the patients that remained low grade. Genome wide DNA methylation levels of recurrent samples were lower than that of initial samples. This difference is explained by the increased number of high grades at recurrence, since near identical DNA methylation levels were observed in samples that remained low grade. Analysis of CNAs revealed chromosomal arms that were more frequently altered in high grade samples. Univariate analysis showed that losses in 3p, 9p, 10q, 13q and 14q were associated with poor OS. More than 800 differentially expressed genes between initial and recurrent tumor samples were found. Chromosomal enrichment analysis revealed a locus on chromosome 6 enriched with histone genes, to be significantly up-regulated over time. Conclusion Longitudinal methylation profiling of IDH-mutant astrocytoma reveals a shift towards a higher grade at tumor recurrence coinciding with reduced DNA methylation levels, and increased frequency of CNAs. Longitudinal expression analysis showed changes in expression of >800 genes, including an up-regulated locus enriched with histone genes. Further integrative analyses are ongoing and will be reported at the meeting.

Background Glioblastoma is the most prevalent and severe type of malignant brain tumor in adults. Although the genetic make-up initiating glioblastoma is increasingly better understood, a better understanding in the mechanisms that drive its evolution, heterogeneity and therapy resistance may reveal new directions for therapy development. To get better insights into glioblastoma evolution, we analyzed and deconvoluted transcriptomes of primary and recurrent glioblastoma resections. Material and Methods Matching primary and secondary resections from n=185 uniformly treated glioblastoma patients were collected as part of EORTC Study 1542 and RNA-sequenced. Data was extended with pairs from n=51 patients from the GLASS study. The datasets were subjected to differential and deconvolution analysis using in-house algorithms. Results When projecting the tumor samples into a reduced Glioblastoma Intrinsic Transcriptional Subtype space, visualization of transitions indicated that the CL subtype switches most often. As we found no more transitions from MES to other subtypes than to be expected by chance, we concluded that MES is an end-state. On average, tumor purity percentages decreased from ~67% to ~50%, mostly due to an increase in macrophages/microglia. Differential expression analysis was performed with correction for the fraction of non-malignant cells. While expression of glioblastoma associated oncogenes did not change significantly over time, marker genes for macrophages/microglia, neurons and oligodendrocytes were up-regulated whereas endothelial cell markers were down-regulated. A cluster of ~30 extracellular matrix associated (ECM) genes increased significantly over time. Single cell RNA-seq, IF-staining and RNA-ish indicated the signature is most strongly expressed near intra-tumoral vessels. Since endothelial marker genes were down-regulated over time, this suggests a form of prognostic vessel progression with a representative transcriptome signature. Conclusion Using a large cohort and validation set of uniformly treated patients, we demonstrate how the glioblastoma transcriptome changes over time with in particular changes the composition of the tumor and its environment. The tumor purity decrease over times suggests a more invasive phenotype or recruitment of non-malignant cells or a combination of both. A post-progression increase in expression of ECM-associated genes expressed near blood vessels was associated with poor survival. Concluding, while no consistent path for transcriptional evolution of tumor cells was observed other than transitions to the MES subtype, glioblastoma becomes more aggressive in time by (re-)organizing its environment.

Background IDH1/2 wildtype (IDHwt) glioma WHO grade 2 and 3 patients with pTERT mutation and/or EGFR amplification and/or +7/−10 chromosome gain/loss have a similar overall survival time as IDHwt glioblastoma patients, and are both considered glioblastoma IDHwt according to the WHO 2021 classification. However, differences in seizure onset have been observed. This study aimed to compare the course of epilepsy in the two glioblastoma subtypes. Material and Methods We analyzed epilepsy data of an existing cohort including IDHwt histologically lower-grade glioma WHO grade 2 and 3 with molecular glioblastoma-like profile (IDHwt hLGG) and IDHwt glioblastoma patients. Primary outcome was the incidence proportion of epilepsy during the disease course. Major secondary outcomes included onset of epilepsy, number of seizure days and antiepileptic drug (AED) polytherapy. Results Out of 254 patients, 78% (50/64) IDHwt hLGG and 68% (129/190) IDHwt glioblastoma patients developed epilepsy during the disease course (p=0.121). Epilepsy onset before histopathological diagnosis occurred more frequently in IDHwt hLGG compared to IDHwt glioblastoma patients (90% versus 60%, p Conclusion Although the incidence proportion of epilepsy during the entire disease course is similar, IDHwt hLGG patients show a significantly higher incidence of epilepsy before diagnosis and a significantly longer median time between first seizure and diagnosis compared to IDHwt glioblastoma patients, indicating a distinct clinical course.

BACKGROUND Temozolomide (TMZ) efficacy in high-grade glioma is related to O 6-methylguanine DNA methyltransferase promoter (MGMTp) methylation. We compared the prognostic and predictive effect of MGMTp between DNA methylation profiling (the MGMT-STP27 model) and quantitative methylation specific polymerase chain reaction (qMS-PCR) in isocitrate dehydrogenase 1 and 2 (IDH1/2) mutant (mt) anaplastic astrocytoma patients. MATERIAL AND METHODS The 2x2 factorial design phase III CATNON trial randomized 751 adult patients with newly diagnosed 1p/19q non-codeleted anaplastic glioma to 59.4 Gy radiotherapy (RT), RT with concurrent TMZ, RT with 12 cycles of adjuvant TMZ, or RT with concurrent and adjuvant TMZ. MGMTp methylation status was assessed with the MGMT-STP27 model using 850k EPIC data, and qMS-PCR. IDH1/2 mutation status was determined with a next-generation sequencing panel. Overall survival (OS) was measured from date of randomization. RESULTS We identified 444 IDH1/2mt anaplastic astrocytoma patients of which MGMT-STP27 data was available for 440 patients (99.1%), qMS-PCR data for 361 patients (81.3%), and both for 357 patients (80.4%). MGMTp was methylated in 365 patients (83.0%) for the MGMT-STP27 model, and 168 patients (46.5%) for qMS-PCR. The agreement between the MGMT-STP27 model and qMS-PCR is 59.9% with a Cohen’s Kappa score of 0.229. At database lock, 289 patients with MGMT-STP27 data were still alive and 236 patients with qMS-PCR data. The median OS of MGMTp methylated glioma patients was 9.1 yrs [95 % confidence interval (CI) 7.5-not reached] for the MGMT-STP27 model, and not reached [95 % CI 9.1-not reached] for the qMS-PCR data. For MGMTp unmethylated glioma patients, the median OS was 6.9 yrs [95% CI 6.2-not reached] for the MGMT-STP27 model, and 6.8 yrs [95% CI 6.2–9.7] for the qMS-PCR data. The hazard ratio (HR) for OS based on MGMTp methylation was 0.88 [95% CI 0.58–1.31] for the MGMT-STP27 data, and 0.72 [95% CI 0.50–1.03]) for the qMS-PCR data. The HR for OS after RT with any TMZ vs RT alone for the MGMT-STP27 model was 0.53 [95% CI 0.37–0.78] for MGMTp methylated, and 0.54 [95% CI 0.25–1.18] for MGMTp unmethylated glioma patients; and for the MS-PCR data was 0.34 [95% CI 0.19–0.61] for MGMTp methylated, and 0.53 [95% CI 0.33–0.85] for MGMTp unmethylated glioma patients. CONCLUSION MGMTp methylation, regardless of assay, was neither prognostic nor predictive for outcome to temozolomide in IDH1/2mt anaplastic astrocytoma patients.

INTRODUCTION The search for effective therapies for gliomas is progressively moving towards patient-specific medicine. In order to test patient-tailored therapies, it is vital to develop protocols for reliable establishment of patient-derived glioma cultures. We present a method for reliable culture establishment, with a 95% success rate in 114 consecutive high-grade samples. METHODS Cell cultures were established from either traditionally-resected tumor tissue or ultrasonic surgical aspirator (CUSA) derived tissue fragments, and expanded in serum-free culture, with selection of astrocytic populations if required. Cultures were started from single cells or small tumor fragments of 0.5-3mm (3D). Whole exome and RNA sequencing were carried out with the Illumina Novaseq and HiSeq platforms. Methylation profiling was performed with the Infinium MethylationEPIC array. Cultures and tumors were compared through analysis of single nucleotide polymorphisms and copy number profiles with the Infinium Global Screening Array. Intra-tumoral heterogeneity in cultures was investigated with single-cell transcriptomic sequencing (SORT-seq). We studied tumor-initiating potential by orthotopic injection of cultures in NOD-SCID mice. RESULTS Cultures started from single cells were established from CUSA material more efficiently (92%) than from traditional resection material (70%). 3D-derived cultures had a higher overall efficiency (95% for CUSA, 85% for traditional resection material). We confirmed high concordance in driver mutations, copy number and methylation profiles between tumors and derived cultures. Transcriptomics analysis, comparing tumors and derived cultures, revealed high consistency in gene expression distribution as demonstrated by correlation analysis (r=0.88). Singe-cell RNA-seq shows increased heterogeneity in CUSA derived-cultures, and decreased heterogeneity with passaging over time. Cultures faithfully produce tumors after orthotopic injection in NOD-SCID mice. CONCLUSION We present a highly successful method for the establishment of glioma cultures from patient material, with CUSA-derived cultures revealing greater heterogeneity. Cultures faithfully represent important molecular characteristics of parental tumors and can be used to test potential therapies in vitro.

_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 cm 3) 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 cm 3 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.

BACKGROUND Oligodendroglioma (OD) is defined by the presence of both IDH1/2 mutation and 1p/19q codeletion. Although prognosis of OD patients is relatively favorable, tumours usually relapse and often evolve to a higher malignancy grade, with some acquiring the treatment induced hypermutated phenotype. To better understand how these tumours evolve in time, we examined the molecular differences between matched primary and recurrent ODs. MATERIAL AND METHODS We identified 21 patients who underwent surgery at least twice [male: 11, female: 10, median age: 44 years (31–66)]. Clinical data were available for 14/21 patients: 5/14 received a treatment between resections [4 radiotherapy, 1 radiotherapy followed by PCV chemotherapy]; median time from the first to the second surgery was 71.5 months (12–158). Whole genome DNA-methylation analysis was performed using Illumina’s MethylationEPIC ‘850K’ BeadChip. Results were evaluated using the Molecularneuropathology.org platform (version 3.1.5) and in R. RESULTS Most samples were WHO grade 2 ODs [14, 10 and 1 tumours in first, second and third resection group, respectively]; WHO grade 3 was found in 6, 10 and 3 tumours in first, second and third resection, respectively; in 4 patients the tumour showed malignant progression from grade 2 to 3. Most ODs exhibited an IDH1 R132H mutation [17/21 patients]; in no cases was IDH1/2 mutation lost during progression. DNA methylation analysis was successfully performed in 41/45 cases [primary OD: 17, recurrent OD: 24] for a total of 18 matched pairs. 37 samples were assigned to the “IDH mutant glioma, subclass 1p/19q codeleted OD”; the remaining 4 were assigned to various other methylation classes but CNV (copy number variation) analysis confirmed the 1p19q codeletion in all samples. Recurrent tumours exhibited de novo loss of chromosome 4 in 3/24 cases (12.5%) and loss of chromosome 13 in 3/24 cases (12.5%). In unsupervised analysis of the 1000 most variable CpG sites, samples from the same patient clustered together. This indicates that the inter-tumour variability is greater than the intra-, temporal- or grading variability between tumours. There were no overt differences in DNA methylation levels between the primary and matched recurrent OD. However, lower genome wide DNA methylation levels were observed in tumours that dedifferentiated to grade 3 ODs compared to those of grade 2, indicating that DNA demethylation is associated to higher malignancy grade. CONCLUSION DNA methylation analysis in a cohort of primary and recurrent oligodendrogliomas highlights the genomic and epi-genetic changes that are acquired at tumour progression. We are currently expanding the cohort and collecting/integrating the clinical data to better explore the evolution of recurrent ODs.

BACKGROUND Delay in cancer care may adversely affect emotional distress, treatment outcome and survival. Optimal timings in multidisciplinary glioblastoma care are a matter of debate and clear national guidelines only exist for time to neurosurgery. We evaluated the between-hospital variation in timings to neurosurgery and adjuvant radiotherapy and chemotherapy in newly diagnosed glioblastoma patients in the Netherlands. MATERIAL AND METHODS Data were obtained from the nation-wide Dutch Brain Tumor Registry between 2014 and 2018. All adult patients with glioblastoma were included, covering all 18 neurosurgical hospitals, 28 radiotherapy hospitals, and 33 oncology hospitals. Long time-to-surgery (TTS) was defined as >3 weeks from the date of first brain tumor diagnosis to surgery, long time-to-radiotherapy (TTR) as either >4 or >6 weeks after surgery, and long time-to-chemotherapy (TTC) as either >4 or >6 weeks after completion of radiotherapy. Between-hospital variation in standardized rate of long timings was analyzed in funnel plots after case-mix correction. RESULTS A total of 4203 patients were included. Median TTS was 20 days and 52.4% of patients underwent surgery within 3 weeks. Median TTR was 20 days and 24.6% of patients started radiotherapy within 4 weeks and 84.2% within 6 weeks after surgery. Median TTC was 28 days and 62.6% of patients received chemotherapy within 4 weeks and 91.8% within 6 weeks after radiotherapy. After case-mix correction, three (16.7%) neurosurgical hospitals had significantly more patients with longer than expected TTS. Three (10.7%) and one (3.6%) radiotherapy hospitals had significantly more patients with longer than expected TTR for >4 and >6 weeks, respectively. In seven (21.2%) chemotherapy hospitals, significantly less patients with TTC >4 weeks were observed than expected. In four (12.1%) chemotherapy hospitals, significantly more patients with TTC >4 weeks were observed than expected. CONCLUSION Between-hospital variation in timings to multidisciplinary treatment was observed in glioblastoma care in the Netherlands. A substantial percentage of patients experienced timings longer than anticipated.

BACKGROUND Over the past years, increasing worldwide attention towards centralization of complex cancer care has been pursued as higher volume centers have shown improved outcomes. Changes in distribution of care and the impact on travel distance in glioblastoma patients have not been determined yet. In this study, we determine trends in distribution of glioblastoma care in the Netherlands over the last three decades and assess whether the observed trends affected travel distance for individual patients. MATERIAL AND METHODS Data were obtained from the Dutch Brain Tumor Registry from 1989 to 2018. All glioblastoma patients (≥18 years) were included for analysis. Patients, neurosurgical centers and radiotherapy centers were geocoded. Data were analyzed in six time intervals of 5 years. High volume hospitals were defined as >50 cases per year. Travel distance was examined in two categories, ≤60km and >60km respectively. Trend analyses for proportions were used to analyze hospital volume changes and travel distances. RESULTS A total of 16.477 glioblastoma patients were registered, with an annual increase from 203 patients in 1989 to 917 patients in 2018. Neurosurgical centers increased from 16 to 17 and for radiotherapy from 19 to 22 centers between 1989–1993 and 2014–2018. Mean neurosurgical- and radiotherapy center volumes increased from 12 to 39 (P=0.025) and 7 to 27 (P=0.025) patients per hospital per year from 1989–1993 to 2014–2018. High volume neurosurgical centers were observed since 2004, and an increased number of patients were treated in these centers, 27.8%, 52.6% and 64.1% in the time periods 2004–2008, 2009–2013, and 2014–2018 (P60km to the neurosurgical center reduced from 15.8% to 13.2% (P=0.033). Travel distance >60km to the radiotherapy center did not reduce significantly (10.4% to 8.8%, P=0.601). CONCLUSION An increasing number of glioblastoma patients were differentially treated in high volume neurosurgery and radiotherapy centers. The observation that this did not translate into increased travel distances, indicates accessible specialized Neuro-Oncology care for glioblastoma patients in The Netherlands.

INTRODUCTION Postponing adjuvant treatment after resection in IDH-mutated low-grade glioma (LGG) seems appropriate in selected patients. Here we describe the clinical course of these patients during post-resection active monitoring with MRI scans, as literature on this topic is scarce. METHODS We included IDH-mutated LGG patients that underwent surgery between 2003–2016, with clinical follow-up including MR scans available. Samples were classified according to the 2016 WHO classification. We collected baseline clinical information, estimates of seizure control during follow-up, longitudinal evolution of post-resection LGG volumes when adjuvant therapy was postponed, and the clinicians’ reasons to either start immediate adjuvant treatment or postpone treatment, and reasons to initiate adjuvant treatment in later stage. We compared patients that underwent immediate postsurgical adjuvant treatment with patients that underwent active MRI monitoring until further progression. RESULTS AND CONCLUSIONS 197 patients were included, of which 108 were IDH-mutated astrocytomas, and 89 were oligodendrogliomas. Of these, 97 patients were actively followed with MRI scans, and 100 patients underwent immediate adjuvant therapy after initial surgery. Patients that received immediate adjuvant therapy were older and had less extensive resections than patients wherein adjuvant treatment was postponed (median age 45 years vs. 37 years, P< 0.001; median postoperative tumor volume 31cm3 vs 2.68cm3, P< 0.001). The duration of the active surveillance period in the patients wherein adjuvant treatment was postponed correlated with the extent of resection, with shorter time until adjuvant treatment in case of larger postoperative tumor volumes. All LGG lesions showed continuous growth during the active surveillance period, with median growth rates of 3mm/year for oligodendrogliomas and 4mm/year in IDH- mutated astrocytomas (P = 0.0093). Seizures were well controlled, with the majority of patients using only one anti-epileptic drug. The majority of patients stayed asymptomatic during follow-up, and the reason to initiate further adjuvant treatment was always radiological growth.

Background The efficacy of novel targeted therapies is often tested at the time of tumor recurrence. However, for glioblastoma (GBM) patients, surgical resections at recurrence are performed only in a minority of patients; therefore, molecular data are predominantly derived from the initial tumor. Molecular data of the initial tumor for patient selection into personalized medicine trials can therefore be used only when the specific genetic change is retained in the recurrent tumor. Methods In this study we determined whether EGFR amplification and expression of the most common mutation in GBMs (EGFRvIII) is retained at tumor recurrence. Because retention of genetic changes may be dependent on the initial treatment, we only used a cohort of GBM samples that were uniformly treated according to the current standard of care (ie, chemo-irradiation with temozolomide). Results Our data show that, in spite of some quantitative differences, the EGFR amplification status remains stable in the majority (84%) of tumors evaluated. EGFRvIII expression remained similar in 79% of GBMs. However, within the tumors expressing EGFRvIII at initial diagnosis, approximately one-half lose their EGFRvIII expression at tumor recurrence. Conclusions The relative stability of EGFR amplification indicates that molecular data obtained in the primary tumor can be used to predict the EGFR status of the recurrent tumor, but care should be taken in extrapolating EGFRvIII expression from the primary tumor, particularly when expressed at first diagnosis.

BACKGROUND Between 1995 and 2002 the EORTC Brain Tumor Group conducted a prospective phase III study on adjuvant procarbazine, CCNU and vincristine (PCV) chemotherapy in anaplastic oligodendroglioma (AOD). A mature follow-up presented in 2012 showed survival benefit of the addition of PCV, in particular in 1p/19q co-deleted tumors and tumors with MGMT promoter methylation. We now present very long term follow-up. MATERIALS AND METHODS Patients were eligible if locally diagnosed with a newly diagnosed AOD. They were randomized between radiotherapy (RT, 33 x 1.8 Gy) and the same RT followed by 6 cycles PCV (RT/PCV). Primary endpoints were overall survival (OS) and progression free survival (PFS). 1p/19q status (FISH) was determined in 300 patient. Kaplan- Meier technique and Cox modeling were used for long term survival analysis. Primary analyses were adjusted for known prognostic factors. For other analyses no adjustment was performed. RESULTS With 368 patients included, a median follow-up of 18.4 years and 307 (83%) survival events, median and 20-year survival after RT/PCV versus RT alone were 42.3 mo and 16.8% vs 30.6 months and 10.1% (HR 0.78; 95% CI (0.63, 0.98), adjusted p=0.06). Eighty patients were 1p/19q codel of which 26 (33%) were still alive, in this subgroup median and 20-year survival after RT/PCV versus RT alone were 14 years and 37.1% versus 9.3 years and 13.6% (HR 0.60, 95% CI (0.35, 1.03), unadjusted p=0.06). Twenty year PFS in 1p/19q codel was 31.3% in RT/PCV treated patients and 10.8% in RT only treated patients (HR 0.49, 95% CI (0.29, 0.83), unadjusted p=0.007). In the 1p/19q codel subgroup age, WHO PS and necrosis at pathology were identified to be of independent prognostic value for OS. CONCLUSION This long term analysis confirms the earlier conclusions and provides data on long term survival in this patient group. In 1p/19q codel patients treated with RT/PCV, the 20-year PFS and OS rates are 31% and 37% respectively.

BACKGROUND Despite considerable scientific efforts, endogenous in vitro isocitrate dehydrogenase (IDH)-mutated glioma models remain scarce. Availability of these models is key to understanding underlying molecular mechanisms and vital for the development of new therapeutic interventions. We established and characterized a set of seven cell lines derived from IDH1-mutated gliomas and utilized them to investigate IDH-mutant glioma drug-response in vitro. MATERIAL AND METHODS Fresh tumor material was collected directly from the operating room, mechanically and enzymatically dissociated and cultured under serum-free culture conditions. IDH mutation status was verified at several passages with Sanger sequencing, as well as with whole exome sequencing. D-2-hydroxyglutarate (D2-HG) levels were determined with mass-spectrometry. Genome-wide methylation profiling was performed using the Infinium MethylationEpic BeadChip array. Cell viability was measured using the ATP-based CellTiter-Glo assay. Cell proliferation was determined through cell counting. Drug screens were performed with an FDA-approved anti-cancer drug set from the NIH as well as two IDH-mutant specific inhibitors. RESULTS Over the last decade our lab processed over 800 glioma samples from all WHO grades and IDH mutational status. Optimisation of culture conditions led to the establishment of seven IDH1-mutant glioma cell lines. The IDH1-mutation was present during all tested passages in these cell lines. Whole exome sequencing showed a high concordance between tumor and cell lines with regard to driver gene mutations. Similarly, copy-number changes based on genome-wide methylation data also show a close resemblance between the parental tumor and resulting cell lines. The Heidelberg methylation profiler classified each cell line and its parental tumor as IDH mutant glioma. When exposed to an IDH1-mutant specific inhibitor, all cell lines showed a concentration-dependent decrease of D2-HG levels. The inhibitors showed little to no effect on viability up to 10uM during 8 days, however, long term treatment up to 4 weeks revealed a decrease in cell doubling time in 2 of 6 cultures. Drug screen results either alone or in combination with an IDH1-mutant specific inhibitor identified several interesting candidates that are currently followed up in additional experiments. CONCLUSION We established a unique set of patient-derived IDH1-mutant glioma cell lines that closely resemble their respective tumors and can be used to identify new therapeutic strategies.

Glial tumors consist of a heterogeneous group of primary CNS neoplasms. While the clinical outcome of these tumors varies substantially between different grades (World Health Organization [WHO] grades I–IV), only grade I tumors can be treated curatively.1 In the last decade, substantial effort has been put into characterizing the pathogenic mechanisms underlying this complex group of glial cell–derived tumors. These efforts have led to the establishment of several molecular subclasses of glioma, which differentiate types of gliomas based on their intrinsic molecular distinctions and similarities, rather than the conventional characteristics used for histology-based grading.2–5 Indeed, gene expression–based clustering of glioma from several grades within the WHO grading system proved more predictive of survival than the histological classification.4, 6 Thus far, however, this gain of insight has not led to improved clinical outcome for patients, since there are no specific treatment strategies designed to target specific subtypes of glioma as of yet. Consequently, the development of preclinical models that accurately reflect the molecular heterogeneity of glioma is imperative for both translationally relevant drug screening programs and the advancement of patient-tailored treatment options. Commercially available cell cultures of glioma have been demonstrated to poorly mimic the molecular aberrations found in patient samples.7,8 Furthermore, the xenograft models derived from these cultures do not sufficiently recapitulate the histological hallmarks of glioma.9 With the advent of the cancer stem cell hypothesis, several groups have implemented serum-free (SF) cell culture regimens, originally developed for neural stem cell propagation, to establish glioma stem-like cell (GSC) cultures from fresh tumor tissue.10,11 By analyzing gene expression profiles of GSCs and serum supplemented (SS) cultures, a distinct separation between the aforementioned and the parental tumors was revealed by unsupervised clustering.7 Several groups have reported GSC cultures to be superior with regard to retaining the original patient gene expression signature and histological phenotype in xenografts.7,11,12 This has led to a wide variety of applications for these cell culture assays, ranging from inquiries into fundamental hypotheses13,14 to preclinical testing of novel agents.15–17 Several contradicting publications have since been published on the optimal cell culture methodology,18,19 mandatory molecular aberrations for successful propagation,20–22 and positive selection for gene expression signatures related to specific molecular subtypes in vitro.14, 23 Most of these previous publications have focused on the characterization of successfully propagated specimens of glioblastoma multiforme (GBM; WHO grade IV) from adults. Reports on the culture success of grade II and grade III gliomas are sparse.20,24 Therefore, we undertook a characterization study of a large cohort (N = 261), which addresses the distribution of glioma from all histological entities for the outcome of GSC culture attempt. Within equal WHO grades, correlations between cell culture outcome and patient overall survival were assessed. Tumor samples of both successful and unsuccessful cultures (n = 46 in total) were also subjected to molecular analysis, and a number of molecular traits that influence cell culture success rate were identified, as well as genes that may play a role in this process. These results emphasize the need for, and provide leads to, the development of improved culture protocols supporting growth of all subtypes of glioma. This is essential for implementation of this model in drug screening programs for personalized treatment strategies.

BACKGROUND: Depatux-M is an antibody-drug-conjugate consisting of an antibody (ABT-806) specific to the activated conformation of EGFR bound to the toxin monomethylauristatin-F. In the primary analysis on EORTC 1410 we reported a trend (p = 0.06) towards improved overall survival (OS) in patients with EGFR-amplified (amp) recurrent glioblastoma treated with Depatux-M in combination with temozolomide. METHODS: Eligible were patients with centrally confirmed EGFRamp glioblastoma at 1st recurrence after temozolomide chemo-irradiation. Patients were randomized to either a) Depatux-M 1.0 mg/kg every 2 weeks intravenously, or b) the same treatment combined with temozolomide 150–200 mg/m2 day 1–5 every 4 weeks, or c) either lomustine or temozolomide (TMZ/LOM) depending on the time of relapse. Primary endpoint was OS. Pharmacokinetic sampling was part of the study design, all samples were used to calculate the Depatux-M average concentration during course 1 (CavgC1). The level of EGFRamp was re-analysed using next generation sequencing. RESULTS: In February 2018, an updated OS comparison performed after 220 observed deaths of Depatux-M in combination with TMZ versus TMZ/LOM using log-rank test and cox models stratified by stratification factors at randomization showed a HR of 0.68 (95%CI [0.48, 0.95]; p = 0.024) and 1-year OS rates of 40% versus 28%. In multivariate analysis CavgC1 was a significant predictor for OS (HR 0.96, 95% CI [0.93, 0.98], p = 0.0013). In Depatux-M treated patients, EGFR status (high vs low level amplification) did not correlate with OS. At the meeting the follow-up from Aug 2018 will be presented, obtained more than 24 months after the end of accrual. CONCLUSION: This updated OS analysis of Depatux-M in combination with temozolomide confirmed the OS improvement in EGFRamp recurrent glioblastoma. In Depatux-M treated patients, higher drug levels during course 1 were associated with improved OS, but high levels of EGFR amplification at first diagnosis were not.

BACKGROUND: EGFR tyrosine kinase inhibitors (TKIs) have thus-far not shown clinical benefit for glioma patients, which is in stark contrast to the benefit observed for patients with EGFR-mutated pulmonary adenocarcinomas (PA). We performed functional analysis on various EGFR-mutation constructs to better understand the differential sensitivity to EGFR TKIs. METHODS: Response to EGFR TKIs was monitored using high-throughput automated imaging analysis, western blot, immunofluorescence, RT-QPCR, and immunoprecipitation-mass spectrometry (IP-MS). RESULTS: Addition of TKIs to cells ectopically expressing EGFR mutation constructs resulted in a rapid and strong formation of EGFR-protein aggregates. However, aggregates were found only cells expressing mutations common to PAs (n=3 mutations tested) but not in mutations common to gliomas (n=4) or those harboring secondary resistance mutations (n=1). Moreover, aggregates were only observed in TKIs with proven clinical benefit (erlotinib, gefitinib, dacomitinib or osimertinib) and not with a drug with no such benefit (lapatinib). We find a high concordance between the IC50 for viability of 13 cell lines harboring endogenous EGFR mutations (n=12 different mutations) and the IC50 of mutations to form protein aggregates. Patients harboring mutations that are sensitive to aggregate formation (IC50 < 500 nM) had significantly longer time to progression (median survival 7.5 vs 13 months, HR 0.25, P=0.012). These data demonstrate that formation of aggregates predicts response to EGFR TKIs in patients. The aggregates are formed, predominantly in the nucleus, first by a dephosphorylation of EGFR after which specific mutations and drugs both affect the (inactive) conformation state of EGFR. Such a state renders the protein more prone to aggregate formation and subsequent complete inactivation. CONCLUSION: Protein aggregation predicts response to EGFR TKIs. Since these aggregates inactivate all functions of the protein, the absence of aggregate formation with glioma specific mutations in EGFR explain why these tumors are insensitive to EGFR TKIs.

In multiple tumor types, prediction of response to immune therapies relate to the presence, distribution and activation state of tumor infiltrating lymphocytes (TILs). Little is known, however, about the immune contexture of TILs in gliomas. To this end, we assessed whether low and high-grade glioma (LGG and HGG) differ with respect to number, location and tumor reactivity of TILs; as well as expression of molecules involved in the trafficking and activation of T cells. TILs were collected from glioma samples and CD3 and CD8 T cells were quantified by flow cytometry (n=10) and immunofluorescence (n=35). Neo-antigen load and expression of Cancer Germline Antigens (CGAs) were assessed using whole exome sequencing and RNA-seq. TIL-derived DNA was sequenced and the variable domain of the TCRβ chain was classified according to IMGT nomenclature (n=5). QPCR was used to determine expression of T cell-related genes (n=20). T cell numbers were significantly lower in LGG. In HGG, CD8 T cells migrated into tumor tissue, whereas in LGG, they mainly remained in close vicinity to blood vessels. Although HGG had significantly more genetic changes, the number of neo-epitopes was only 2 in both tumor types. We also did not observe a difference in the expression of CGAs nor in dominant TCR-Vb gene usage. The low number and perivascular location of CD8 T cells in LGG was accompanied by a low expression of chemo-attractants CXCL9 and CXCL10, adhesion molecule ICAM1 and CD8 T cell effector molecule GZMK. LGG have lower numbers of TILs compared to HGG, potentially linked to decreased T cell extravasation and activation. We have found no evidence for distinct tumor reactivity of T cells in either tumor type. Our data argue that LGG would require sensitization to enhance numbers and trafficking of CD8 T cells prior to immune therapies, such as checkpoint inhibitors.

INTRODUCTION: Current efforts to improve patient survival in recurrent glioblastomas (GBMs) are often based on targeting the tumors acquired genetic changes. However, most molecular data is derived from the initial tumor: resections of recurrent GBMs are seldom performed. This study aims to assess whether genetic traits of initial GBMs are still present at recurrence. METHODS: DNA/RNA was isolated from pairs of initial and recurrent Stupp-treated GBM tumor samples (FFPE) and sequenced on a panel of 365 cancer genes. MGMT promotor methylation was determined by MS-PCR, EGFRvIII by RT-PCR and TERT-promotor mutations by SNaPshot. RESULTS: 276 patients from ten medical centers in six countries were identified with median age of 54.1 years. Median survival was 23.7 months, and median time to second surgery 13.0 months. Only 10 of 186 sequenced matched tumor pairs were IDH-mutated (5.4%). Overall, genetic traits of initial GBMs were stable with a median retention rate of coding mutations of 81.8%. Similarly, copy number changes (CNVkit/GISTIC) generally were retained at tumor recurrence. However, the retention rate was also ~80% in all of the major GBM driver pathways (TP53 signaling, RAS-RAF-MEK-ERK, RTK signaling). This is important as decreases 20% of loss of a marker requires a doubling of the number of included patients to achieve a similar power (assuming an objective response rate of 40% as a positive outcome). One noticeable change was a loss of TERT-promoter mutations in 7.5% of recurrent samples. Only four tumors were hypermutated (> 100 coding mutations) at recurrence, though more samples (n=12) acquired mutations in MSH2 and MSH6. Only one EGFR-amplified tumor was hypermutated. CONCLUSION: EGFR-amplified GBMs seldom transform into hypermutated tumors which suggests immune-checkpoint inhibitors have limited clinical use in recurrent GBMs. Re-sampling should be considered prior to inclusion in targeted therapy trials to ensure proper patient selection.

INTRODUCTION: Several studies reported a correlation between the anatomic location of gliomas and the genetic background of the tumor. As such, tumor location may contribute to pre-surgical clinical decision-making. Current evidence is mainly derived from smaller series and report single genes or copy numbers, but not the integrated WHO 2016 classification. Our purpose was to assess the location distribution of different WHO 2016 glioma subgroups in a consecutive series of molecularly defined grade II glioma and to create location heatmaps of the low grade glioma molecular subtypes. METHODS: 205 adult patients with a grade II supratentorial glioma diagnosed between 2003–2016 were included in this study (100 IDH mutated astrocytoma, 85 IDH mutated and 1p19q co-deleted oligodendroglioma, and 20 IDH wildtype astrocytoma). All patients were classified according to the WHO 2016 criteria using a dedicated Next-Generation-Sequencing panel. Tumor volume and location were assessed with semi-automatic software on T2-weighted images. All volumes of interest were mapped to a standard brain using affine registration. Location heatmaps were created for each WHO 2016 glioma subgroup by overlaying segmentations on a standard brain. RESULTS: Our location maps confirmed earlier observations that different molecular subtypes of grade II glioma have a different spatial distribution: most IDH mutated 1p19q co-deleted oligodendrogliomas were located in the frontal lobes and cortex, while IDH mutated astrocytomas were more frequently located in the frontotemporal and insular region. IDH wildtype astrocytomas were predominantly located in the basal ganglia and temporal lobes. Our observations were significant as confirmed with voxel-based Fisher’s exact tests (figures will be shown at SNO). CONCLUSION: Each WHO 2016 glioma subgroup displayed a distinct pattern of spatial distribution. Our observations may contribute to pre-surgical clinical decision-making and to radiogenomics.

Following the trials of bevacizumab for the treatment of glioblastoma multiforme, there has been considerable discussion over whether this drug should be recommended as a standard treatment for this disease. Trial data suggests prolongation of progression-free survival, but no effect on overall survival. Despite these results, tails of Kaplan Meier curves and observations on a case-by-case basis suggest bevacizumab improves overall survival in certain patient subsets. We used TCGA microRNA expression data from tumors resected at first diagnosis of glioblastoma in patients treated with bevacizumab and randomly separated these into training (n = 50) and test (n = 37) groups. Using a cross-validated penalized regression model we identified 8 microRNAs as potential predictors of overall survival in the training set. We created a response score using the total expression of these microRNAs, weighted by regression coefficient. This was dichotomized based on the most prognostic minimum of a density plot of the response scores (Log-rank HR = 0.16, p = 1.2e-5). This was then validated in the test cohort (one-sided log-rank HR = 0.34, p = 0.026). Analysis of the signature using all samples in the TCGA, regardless of treatment received, (n = 562) showed that the response prediction was much less powerful (HR = 0.73, p = 1.6e-3) in this cohort suggesting the signature is specific to bevacizumab. The definition of a bevacizumab responding group was validated using expression of microRNAs from RNA-seq data from patients treated with bevacizumab or CCNU at recurrence in the BELOB trial. This showed that ‘responders’ defined by the microRNA signature survived significantly longer than ‘non-responders’ after treatment with bevacizumab (n= 20, HR = 0.23, p = 0.020) but not after treatment with CCNU (n= 22, HR = 0.35, p = 0.052). In conclusion, we have identified an 8-microRNA signature that predicts overall survival in patients with glioblastoma treated with bevacizumab. This may be useful for identifying patients who are likely to benefit from this agent.

BACKGROUND: Molecular holds promise for an improved classification of glial tumors. We analyzed samples of patients enrolled in EORTC study 26951 on adjuvant PCV in anaplastic oligodendroglial tumors to evaluate 1. The clinical significance of glioma classification with Next Generation Sequencing (NGS); and 2. The prognostic significance of CIC and FUBP mutations. MATERIAL AND METHODS: EORTC 26951 investigated adjuvant PCV in locally diagnosed anaplastic oligodendroglial tumors. NGS was performed on archival formalin fixed paraffin embedded (FFPE) samples using the Ion Torrent Personal Genome Machine for simultaneous detection of mutations in the genes for ATRX, CIC, EGFR, FUBP1, NOTCH1, PTEN, and TP53 and hot spot mutation regions in BRAF, H3F3A, IDH1, IDH2 and PIK3CA as well as imbalances on chromosome 1p, 10q, 7 en 19q. TERT mutations were assessed with PCR. RESULTS: At the time of abstract submission 89 cases have been investigated, more samples are being processed. In 62 tumors an IDH1 mutations was found, in 2 an IDH 2 mutation. Forty-eight tumors (54%) were 1p/19q codeleted, 29 (60%) of these had CIC mutations and 20 (42%) had FUBP mutations. All but one 1p/19q codeleted tumors showed TERT mutations. Ten tumors showed ATRX mutations, 5 also had PTEN mutations. Median OS was 136 months for CIC mutated 1p/19q co-delete tumors, and 95 months for CICwt tumors (p = 0.17); FUBP mutations showed no relation with OS. TERT mutations were found in 60 tumors (67%), OS in TERT mutated but 1p/19q intact tumors was 12 months. IDH mutations, TERT mutations and 1p/19q co-deletion were the most significant prognostic factors, distinguishing between groups with significantly different OS. CONCLUSIONS: The ongoing expansion of this series will show if CIC mutations have prognostic significance. Targeted NGS even on archival FFPE allows a clinically relevant prognostic classification of glioma, while simultaneously identifying relevant druggable targets.

BACKGROUND: EGFR is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumor types have different mutation spectra. It is possible that functional differences underlie this tumor-type specific mutation spectrum. METHODS: We have determined whether specific mutations in EGFR (EGFR, EGFRvIII and EGFR-L858R) have differences in binding partners, differences in downstream pathway activation (gene expression and phosphoproteins), and have functional consequences on cellular growth and migration. RESULTS: Using biotin pulldown and subsequent mass spectrometry we were able to detect mutation specific binding partners for EGFR. Differential binding was confirmed using a proximilty ligation assay and/or Western Blot for the dedicator of cytokinesis 4 (DOCK4), UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), MYC binding protein 2 (MYCBP2) and Smoothelin (SMTN). We also demonstrate that each mutation induces the expression of a specific set of genes, and that each mutation is associated with specific phosphorylation patterns. Finally, we demonstrate using stably expressing cell lines that EGFRvIII and EGFL858R display reduced growth and migration compared to EGFR wildtype expressing cells. CONCLUSION: Our results indicate that there are distinct functional differences between different EGFR mutations. The functional differences between different mutations argue for the development of mutation specific targeted therapies.

BACKGROUND: With the rapid discovery of prognostic and predictive molecular parameters for glioma, the status of histopathology in the diagnostic process should be scrutinized. The present project aimed at the construction of an evidence-based diagnostic algorithm for gliomas. To reach this goal, histological features were scrutinized for their prognostic relevance and acceptable inter-observer variability. In addition, molecular markers should be validated for their prognostic power and possible overlap when simultaneously applied. METHODS: The pathology slides of EORTC trials 26951 and 26882 were scanned for virtual microscopy, an electronic scoring form was created and a panel of six pathologists was formed to independently evaluate the slides. The molecular analysis for IDH1, 1p/19q loss; EGFR amplification; loss of chromosome 10 and 10q, gains of chromosome 7 and hypermethylation of the promoter of MGMT were available for appr. half of cases. The slides were divided in a test (discovery) set (n = 500) and a validation set (n = 300). All follow-up data were available at the EORTC data center in Brussels and the results were statistically tested with the aim to create and validate the diagnostic algorithm. RESULTS: In 66% of cases consensus of overall diagnosis was present. Cox regression analysis of consensus histological features and molecular markers revealed the prognostic significance of the parameters necrosis, cell density, mitotic index, loss 1p/19q and IDH1 mutation. A diagnostic algorithm consisting of relevant (consensus and prognostically significant) histological features and molecular markers was identified by CART analysis and consisted of 2 molecular markers and 1 histological feature. The order of prognostic power was: 1p/19q loss; EGFR amplification; astrocytic morphology, and 4 diagnostic nodes were identified. Validation of the nodes in the validation set yielded 4 significantly different survival curves (Overall Wald test p < 0.0001). CONCLUSION: we succeeded in the creation of a timely combined histologic and molecular diagnostic algorithm.