Mitochondrial DNA copy number as a prognostic marker is age-dependent in adult glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive form of glioma. GBM frequently displays chromosome (chr) 7 gain, chr 10 loss and/or EGFR amplification (chr7+/chr10-/EGFRamp). Overall survival (OS) is 15 months after treatment. In young adults, IDH1/2 mutations are associated with longer survival. In children, histone H3 mutations portend a dismal prognosis. Novel reliable prognostic markers are needed in GBM. We assessed the prognostic value of mitochondrial DNA (mtDNA) copy number in adult GBM. METHODS: mtDNA copy number was assessed using real-time quantitative PCR in 232 primary GBM. Methylation of POLG and TFAM genes, involved in mtDNA replication, was assessed by bisulfite-pyrosequencing in 44 and 51 cases, respectively. RESULTS: Median age at diagnosis was 56.6 years-old and median OS, 13.3 months. 153/232 GBM (66 %) displayed chr7+/chr10-/EGFRamp, 23 (9.9 %) IDH1/2 mutation, 3 (1.3 %) H3 mutation and 53 (22.8 %) no key genetic alterations. GBM were divided into two groups, "Low" (n = 116) and "High" (n = 116), according to the median mtDNA/nuclear DNA ratio (237.7). There was no significant difference in OS between the two groups. By dividing the whole cohort according to the median age at diagnosis, OS was longer in the "High" vs "Low" subgroup (27.3 vs 15 months, P = .0203) in young adult GBM (n = 117) and longer in the "Low" vs "High" subgroup (14.5 vs 10.2 months, P = .0116) in older adult GBM (n = 115). POLG was highly methylated, whereas TFAM remained unmethylated. CONCLUSION: mtDNA copy number may be a novel prognostic biomarker in GBM, its impact depending on age.

RNA-sequencing of IDH-wild-type glioblastoma with chromothripsis identifies novel gene fusions with potential oncogenic properties.

Glioblastoma (GBM) is the most frequent and most aggressive form of glioma. It is characterized by marked genomic instability, which suggests that chromothripsis (CT) might be involved in GBM initiation. Recently, CT has emerged as an alternative mechanism of cancer development, involving massive chromosome rearrangements in a one-step catastrophic event. The aim of the study was to detect CT in GBM and identify novel gene fusions in CT regions. One hundred and seventy IDH-wild-type GBM were screened for CT patterns using whole-genome single nucleotide polymorphism (SNP) arrays. RNA sequencing was performed in 52 GBM with CT features to identify gene fusions within CT regions. Forty tumors (40/52, 77%) harbored at least one gene fusion within CT regions. We identified 120 candidate gene fusions, 30 of which with potential oncogenic activities. We validated 11 gene fusions, which involved the most recurrent fusion partners (EGFR, SEPT14, VOPP1 and CPM), by RT-PCR and Sanger sequencing. The occurrence of CT points to underlying gene fusions in IDH-wild-type GBM. CT provides exciting new research avenues in this highly aggressive cancer.

Whole genome duplication is an early event leading to aneuploidy in IDH-wild type glioblastoma.

Glioblastoma, the most frequent and lethal form of glioma, displays chromosome instability and recurrent somatic copy number alterations (SCNA). Chromothripsis and whole genome duplication (WGD) have been recently identified in cancer. In the present study, we analyzed SCNA and determine the ploidy pattern in 123 IDH-wild-type glioblastomas, using SNP array data. WGD and chromothripsis events were validated using, respectively, FISH and CTLPScanner. WGD was detected in 11.4% glioblastomas (14/123) and was associated with TP53 mutation (p = 0.0068). It was an early event occurring after the recurrent SCNA observed in diffuse high-grade gliomas. Glioblastomas with WGD were more aneuploid compared to glioblastomas without WGD (p < 0.0001). Chromothripsis occurred in 29.3% glioblastomas (36/123) and mostly affected chromosomes 7, 9 and 12, with amplification of oncogenes (EGFR, MDM2/CDK4), and homozygous deletion of tumor suppressor genes (CDKN2A). There was a significant association between chromothripsis and gene rearrangement at a given locus. WGD is an early genetic event significantly associated to TP53 mutation and leading to chromosome instability and aneuploidy in IDH-wild-type glioblastoma. Chromothripsis recurrently targets oncogenes and tumor suppressor genes that are key players in gliomagenesis and tumor progression. The occurrence of chromothripsis points to underlying gene rearrangements (including gene fusions), potential therapeutic targets in glioblastoma.

Identification of novel recurrent ETV6-IgH fusions in primary central nervous system lymphoma.

Background: Primary central nervous system lymphoma (PCNSL) represents a particular entity within non-Hodgkin lymphomas and is associated with poor outcome. The present study addresses the potential clinical relevance of chimeric transcripts in PCNSL discovered by using RNA sequencing (RNA-seq). Methods: Seventy-two immunocompetent and newly diagnosed PCNSL cases were included in the present study. Among them, 6 were analyzed by RNA-seq to detect new potential fusion transcripts. We confirmed the results in the remaining 66 PCNSL. The gene fusion was validated by fluorescence in situ hybridization (FISH) using formalin-fixed paraffin-embedded (FFPE) samples. We assessed the biological and clinical impact of one new gene fusion. Results: We identified a novel recurrent gene fusion, E26 transformation-specific translocation variant 6-immunoglobulin heavy chain (ETV6-IgH). Overall, ETV6-IgH was found in 13 out of 72 PCNSL (18%). No fusion conserved an intact functional domain of ETV6, and ETV6 was significantly underexpressed at gene level, suggesting an ETV6 haploinsufficiency mechanism. The presence of the gene fusion was also validated by FISH in FFPE samples. Finally, PCNSL samples harboring ETV6-IgH showed a better prognosis in multivariate analysis, P = 0.03, hazard ratio = 0.33, 95% CI = 0.12-0.88. The overall survival at 5 years was 69% for PCNSL harboring ETV6-IgH versus 29% for samples without this gene fusion. Conclusions: ETV6-IgH is a new potential surrogate marker of PCNSL with favorable prognosis with ETV6 haploinsufficiency as a possible mechanism. The potential clinical impact of ETV6-IgH should be validated in larger prospective studies.

BRAF-V600E immunohistochemistry in a large series of glial and glial-neuronal tumors.

INTRODUCTION: Some glial-neuronal tumors (GNT) (pleomorphic xantho-astrocytoma [PXA], ganglioglioma [GG]) display BRAF-V600E mutation, which represents a diagnostic clue to these entities. Targeted therapies against BRAF-V600 protein have shown promising results in GNT. The aim of this study was to assess the utility of BRAF-V600E immunohistochemistry (IHC, clone VE1) in daily practice in a series of 140 glial, and GNT compared to molecular biology (MB) techniques. METHODS: We performed BRAF-V600E IHC on all 140 cases. We used Sanger sequencing and allele-specific quantitative PCR (ASQ-PCR) to detect BRAF-V600E mutation when sufficient amount of materiel was available. RESULTS: BRAF-V600E immunostaining was detected in 29.5% of cases (41/140 cases; 61.5% GG/GC/AGG (32/52), 33% PXA, 6.6% pilocytic astrocytomas). In 47 cases, MB could be performed: Sanger sequencing and ASQ-PCR in 34 cases, ASQ-PCR only in 11 cases, and Sanger sequencing only in two cases. In initial tumors, Sanger sequencing identified BRAF-V600E mutation in 19.5% tumors (seven of 36 tested cases). ASQ-PCR showed mutation in 48.5% tumors (17/35 tested cases). In six cases (5 GG, one PXA), the results were discordant between IHC and MB; the five GG cases were immunopositive for BRAF-V600E but wild type with both MB techniques. In another 7 GG, the percentage of mutated (ganglion) cells was low, and Sanger sequencing failed to detect the mutation, which was detected by IHC and ASQ-PCR. CONCLUSIONS: In tumors with few mutated cells (e.g., GG), anti-BRAF-V600E IHC appears more sensitive than Sanger sequencing. The latter, although considered as the gold standard, is not to be used up-front to detect BRAF mutation in GG. The combination of IHC and ASQ-PCR appears more efficient to appraise the indication of targeted therapies in these glioneuronal tumors.

Chromosome 17p Homodisomy Is Associated With Better Outcome in 1p19q Non-Codeleted and IDH-Mutated Gliomas.

BACKGROUND: The 1p19q non-codeleted gliomas with IDH mutation, defined as "molecular astrocytomas," display frequent TP53 mutations and have an intermediate prognosis. We investigated the prognostic impact of copy number-neutral loss of heterozygosity (CNLOH) in 17p in this population. METHODS: We analyzed 793 gliomas (206 grade II, 377 grade III, and 210 grade IV) by single nucleotide polymorphism array and for TP53 mutations. RESULTS: Homodisomy revealed by CNLOH was observed in 156 cases (19.7%). It was more frequent in astrocytomas and oligoastrocytomas (98/256, 38%) than oligodendrogliomas (28/327, 8.6%; p < .0001) or glioblastoma multiforme (30/210, 14.3%; p < .0001), tightly associated with TP53 mutation (69/71 vs. 20/79; p = 2 × 10(-16)), and mutually exclusive with 1p19q codeletion (1/156 vs. 249/556; p < .0001). In the group of IDH-mutated 1p19q non-codeleted gliomas, CNLOH 17p was associated with longer survival (86.3 vs. 46.2 months; p = .004), particularly in grade III gliomas (overall survival >100 vs. 37.9 months; p = .007). These data were confirmed in an independent dataset from the Cancer Genome Atlas. CONCLUSION: CNLOH 17p is a prognostic marker and further refines the molecular classification of gliomas. IMPLICATIONS FOR PRACTICE: Homodisomy of chromosome 17p (CNLOH 17p) is a frequent feature in IDH-mutated 1p19q non-codeleted gliomas (group 2). It is constantly associated with TP53 mutation. It was found, within this specific molecular group of gliomas (corresponding to molecular astrocytomas), that CNLOH 17p is associated with a much better outcome and may therefore represent an additional prognostic marker to refine the prognostic classification of gliomas.

Severe hydrocephalus caused by diffuse leptomeningeal and neurocutaneous melanocytosis of antenatal onset: a clinical, pathologic, and molecular study of 2 cases.

Diffuse leptomeningeal melanocytosis (DLM) is a rare nevomelanocytic proliferation arising in the meninges. Despite their lack of morphological features of malignancy, these clonal nevomelanocytic cells are capable of extensive invasion and of malignant behavior. When associated with congenital melanocytic nevi, the disorder is named neurocutaneous melanocytosis (NCM). When symptomatic, DLM is usually revealed during childhood, but some cases remain clinically silent. The aim of this study was to analyze melanocytic proliferation in 2 rare and severe cases of isolated DLM and NCM of prenatal onset by neuropathologic and molecular analysis. We performed neuropathologic examination, comparative genomic hybridization arrays, fluorescence in situ hybridization, BRAF and NRAS pyrosequencing in the 2 cases, and next-generation sequencing in the case of isolated DLM. The neuropathologic examination showed diffuse meningeal melanocytic proliferation involving the whole central nervous system with multiple areas of intraneural invasion, associated with large nevi in 1 case. We did not find any chromosomal imbalances. A NRAS(Q61K) mutation was found in the cutaneous and meningeal lesions from the NCM. No mutation was found within a panel of oncogenes including BRAF, NRAS, HRAS, KIT, GNAQ, and GNA11 concerning the isolated DLM. We report 2 exceptional cases of hydrocephalus of prenatal onset related to DLM and NCM. The molecular mechanisms underlying our case of DLM remain unsolved despite the panel of analysis applied.

Detection, Characterization, and Inhibition of FGFR-TACC Fusions in IDH Wild-type Glioma.

PURPOSE: Oncogenic fusions consisting of fibroblast growth factor receptor (FGFR) and TACC are present in a subgroup of glioblastoma (GBM) and other human cancers and have been proposed as new therapeutic targets. We analyzed frequency and molecular features of FGFR-TACC fusions and explored the therapeutic efficacy of inhibiting FGFR kinase in GBM and grade II and III glioma. EXPERIMENTAL DESIGN: Overall, 795 gliomas (584 GBM, 85 grades II and III with wild-type and 126 with IDH1/2 mutation) were screened for FGFR-TACC breakpoints and associated molecular profile. We also analyzed expression of the FGFR3 and TACC3 components of the fusions. The effects of the specific FGFR inhibitor JNJ-42756493 for FGFR3-TACC3-positive glioma were determined in preclinical experiments. Two patients with advanced FGFR3-TACC3-positive GBM received JNJ-42756493 and were assessed for therapeutic response. RESULTS: Three of 85 IDH1/2 wild-type (3.5%) but none of 126 IDH1/2-mutant grade II and III gliomas harbored FGFR3-TACC3 fusions. FGFR-TACC rearrangements were present in 17 of 584 GBM (2.9%). FGFR3-TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. They are mutually exclusive with IDH1/2 mutations and EGFR amplification, whereas they co-occur with CDK4 amplification. JNJ-42756493 inhibited growth of glioma cells harboring FGFR3-TACC3 in vitro and in vivo. The two patients with FGFR3-TACC3 rearrangements who received JNJ-42756493 manifested clinical improvement with stable disease and minor response, respectively. CONCLUSIONS: RT-PCR sequencing is a sensitive and specific method to identify FGFR-TACC-positive patients. FGFR3-TACC3 fusions are associated with uniform intratumor expression of the fusion protein. The clinical response observed in the FGFR3-TACC3-positive patients treated with an FGFR inhibitor supports clinical studies of FGFR inhibition in FGFR-TACC-positive patients.

VEGFA SNP rs2010963 is associated with vascular toxicity in recurrent glioblastomas and longer response to bevacizumab.

Although anti-VEGF therapy is widely used in high-grade gliomas, no predictor of response or toxicity has been reported yet. We investigated here the association of the functional single nucleotide polymorphism (SNP) rs2010963, located in the 5' untranslated terminal region of the VEGFA gene, with survival, response to bevacizumab (BVZ) and vascular toxicity. The rs2010963 was genotyped by Taqman assay in blood DNA from 954 glioma patients with available survival data, including 225 glioblastoma (GBM) patients treated with BVZ. VEGFA plasma levels were assessed by ELISA in 87 patients before treatment. Thrombo-hemorragic adverse events were recorded during BVZ treatment or not, and in an independent population of 92 GBM patients treated with temozolomide. The CC genotype was associated with the occurrence of thrombo-hemorragic events (CC 25 versus CG 13.5 and GG 5.2 %; P = 0.0044) during BVZ. A similar but weaker and non significant trend was observed in patients not receiving BVZ. A CC genotype was associated with higher levels of plasma VEGFA at baseline (107.6 versus 57.50 pg/mL in heterozygotes (CG) and 52.75 pg/mL in GG patients, P = 0.035 and P = 0.028 respectively). The CC genotype tended to be associated to longer PFS when treated with BVZ (P = 0.05), but not when treated with the temozolomide treatment. Our data suggest that the rs2010963 genotype is associated with longer PFS, higher risk of vascular events in recurrent GBM especially treated with BVZ, and higher plasma VEGFA concentration. It may help to identify patients at risk of vascular adverse events during BVZ treatment.

Combined analysis of TERT, EGFR, and IDH status defines distinct prognostic glioblastoma classes.

OBJECTIVE: To identify the prognostic significance of TERT promoter mutations (TERTp-mut) and their associations with common molecular alterations in glioblastomas (GBMs). METHODS: We sequenced the TERTp-mut in DNA from 395 GBMs and analyzed the results with their respective histology, genetic profile (IDH1 mutation, EGFR amplification, CDKN2A homozygous deletion, loss of chromosome 10, TP53 mutation), and overall survival (OS). RESULTS: TERTp-mut were found in 299 of 395 GBMs (75.7%) and were associated with an older age (median 59.6 years for TERTp-mut vs 53.6 years for TERT promoter wild type [TERTp-wt], p < 0.0001). TERTp-mut was an independent factor of poor prognosis (OS = 13.8 vs 18.4 months), in both IDH-mutated (OS = 13.8 vs 37.6 months, p = 0.022) and IDH-wt GBMs (OS = 13.7 vs 17.5 months, p = 0.006). TERTp-mut was associated with IDH-wt, EGFR amplification, CDKN2A deletion, and chromosome 10q loss, but not with MGMT promoter methylation. In the TERTp-wt group, OS was twice longer in EGFR-wt than in EGFR amplification GBMs (OS = 26.6 vs 13.3 months; p = 0.005). In the EGFR-wt group, patients with TERTp-wt had a significantly better outcome (OS = 26.3 vs 12.5 months, p < 0.0001), whereas in the EGFR amplification group, patients with TERTp-mut survived longer (OS = 15.8 vs 13.3 months, p = 0.05). Taken together, the absence of both EGFR amplification and TERTp-mut is associated with longer survival in patients with GBM (26.5 months for patients with IDH-wt, 36.7 months for patients with IDH mutation). CONCLUSIONS: The analysis of TERTp-mut, in combination with EGFR amplification and IDH mutation status, refines the prognostic classification of GBMs.

A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.

INTRODUCTION: Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing. RESULTS: The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results. CONCLUSIONS: The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

Mutational analysis of primary central nervous system lymphoma.

Little is known about the genomic basis of primary central nervous system lymphoma (PCNSL) tumorigenesis. To investigate the mutational profile of PCNSL, we analyzed nine paired tumor and germline DNA samples from PCNSL patients by high throughput exome sequencing. Eight genes of interest have been further investigated by focused resequencing in 28 additional PCNSL tumors to better estimate their incidence. Our study identified recurrent somatic mutations in 37 genes, some involved in key signaling pathways such as NFKB, B cell differentiation and cell cycle control. Focused resequencing in the larger cohort revealed high mutation rates for genes already described as mutated in PCNSL such as MYD88 (38%), CD79B (30%), PIM1 (22%) and TBL1XR1 (19%) and for genes not previously reported to be involved in PCNSL tumorigenesis such as ETV6 (16%), IRF4 (14%), IRF2BP2 (11%) and EBF1 (11%). Of note, only 3 somatically acquired SNVs were annotated in the COSMIC database. Our results demonstrate a high genetic heterogeneity of PCNSL and mutational pattern similarities with extracerebral diffuse large B cell lymphomas, particularly of the activated B-cell (ABC) subtype, suggesting shared underlying biological mechanisms. The present study provides new insights into the mutational profile of PCNSL and potential targets for therapeutic strategies.

IDH mutations: genotype-phenotype correlation and prognostic impact.

IDH1/2 mutation is the most frequent genomic alteration found in gliomas, affecting 40% of these tumors and is one of the earliest alterations occurring in gliomagenesis. We investigated a series of 1305 gliomas and showed that IDH mutation is almost constant in 1p19q codeleted tumors. We found that the distribution of IDH1(R132H) , IDH1(nonR132H) , and IDH2 mutations differed between astrocytic, mixed, and oligodendroglial tumors, with an overrepresentation of IDH2 mutations in oligodendroglial phenotype and an overrepresentation of IDH1(nonR132H) in astrocytic tumors. We stratified grade II and grade III gliomas according to the codeletion of 1p19q and IDH mutation to define three distinct prognostic subgroups: 1p19q and IDH mutated, IDH mutated--which contains mostly TP53 mutated tumors, and none of these alterations. We confirmed that IDH mutation with a hazard ratio = 0.358 is an independent prognostic factor of good outcome. These data refine current knowledge on IDH mutation prognostic impact and genotype-phenotype associations.

Tumor and endothelial cell hybrids participate in glioblastoma vasculature.

BACKGROUND: Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM). Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM. MATERIALS AND METHODS: We used EGFR FISH Probe to detect EGFR amplification and anti-CD31, CD105, VE-cadherin, and vWF to identify endothelial cells. Endothelial and GBM cells were grown separately, labeled with GFP and DsRed lentiviruses, and then cocultured with or without contact. RESULTS: In a subset of GBM tissues, we found that several tumor endothelial cells carry EGFR amplification, characteristic of GBM tumor cells. This observation was reproduced in vitro: when tumor stem cells derived from GBM were grown in the presence of human endothelial cells, a fraction of them acquired endothelial markers (CD31, CD105, VE-cadherin, and vWF). By transduction with GFP and DsRed expressing lentiviral vectors, we demonstrate that this phenomenon is due to cell fusion and not transdifferentiation. CONCLUSION: A fraction of GBM stem cells thus has the capacity to fuse with endothelial cells and the resulting hybrids may participate in tumor microvascular proliferation and in treatment resistance.

An ANOCEF genomic and transcriptomic microarray study of the response to irinotecan and bevacizumab in recurrent glioblastomas.

BACKGROUND: We performed a retrospective study to assess whether the initial molecular characteristics of glioblastomas (GBMs) were associated with the response to the bevacizumab/irinotecan chemotherapy regimen given at recurrence. RESULTS: Comparison of the genomic and gene expression profiles of the responders (n = 12) and nonresponders (n = 13) demonstrated only slight differences and could not identify any robust biomarkers associated with the response. In contrast, a significant association was observed between GBMs molecular subtypes and response rates. GBMs assigned to molecular subtype IGS-18 and to classical subtype had a lower response rate than those assigned to other subtypes. In an independent series of 33 patients, neither EGFR amplification nor CDKN2A deletion (which are frequent in IGS-18 and classical GBMs) was significantly associated with the response rate, suggesting that these two alterations are unlikely to explain the lower response rate of these GBMs molecular subtypes. CONCLUSION: Despite its limited sample size, the present study suggests that comparing the initial molecular profiles of responders and nonresponders might not be an effective strategy to identify biomarkers of the response to bevacizumab given at recurrence. Yet it suggests that the response rate might differ among GBMs molecular subtypes.

Clinical value of chromosome arms 19q and 11p losses in low-grade gliomas.

BACKGROUND: Diffuse low-grade gliomas (LGGs) form a heterogeneous subgroup of gliomas in adults. Chromosome (chr) arms 1p/19q codeletion and IDH mutation have been shown to be closely associated with oligodendroglial phenotype and better prognosis. We sought to identify relevant biomarkers in non 1p/19q codeleted LGGs. METHODS: We characterized a retrospective series of 126 LGGs using genomic arrays, microsatellite analysis, IDH sequencing, MGMT promoter methylation assay, and p53 expression analysis. RESULTS: Our study confirms that 1p/19q codeletion, mutually exclusive with p53 overexpression, was associated with: (i) better prognosis, (ii) oligodendroglial phenotype, (iii) MGMT promoter methylation, and (iv) IDH mutation. Interestingly, 1p/19q codeleted tumors occur in older patients at diagnosis. Our study shows that non 1p/19q codeleted LGGs can be divided in 5 main genomic subgroups: (i) 11p loss, (ii) 19q loss (iii) 7 gain, (iv) 19 gain, and (v) unclassified. In non 1p/19q codeleted LGGs, we demonstrated that (i) 11p loss is associated with astrocytoma phenotype and has an independent negative prognostic value, and (ii) 19q loss diminished the favorable prognostic value of IDH mutation. Our findings were validated in an independent cohort of 98 LGGs. CONCLUSION: Novel genomic entities and biomarkers have been identified in non 1p/19q codeleted LGGs. Our findings may help to stratify non 1p/19q codeleted LGGs, facilitating future individualization of treatment. Further prospective studies are warranted to support our findings.

Deciphering the 8q24.21 association for glioma.

We have previously identified tagSNPs at 8q24.21 influencing glioma risk. We have sought to fine-map the location of the functional basis of this association using data from four genome-wide association studies, comprising a total of 4147 glioma cases and 7435 controls. To improve marker density across the 700 kb region, we imputed genotypes using 1000 Genomes Project data and high-coverage sequencing data generated on 253 individuals. Analysis revealed an imputed low-frequency SNP rs55705857 (P = 2.24 × 10(-38)) which was sufficient to fully capture the 8q24.21 association. Analysis by glioma subtype showed the association with rs55705857 confined to non-glioblastoma multiforme (non-GBM) tumours (P = 1.07 × 10(-67)). Validation of the non-GBM association was shown in three additional datasets (625 non-GBM cases, 2412 controls; P = 1.41 × 10(-28)). In the pooled analysis, the odds ratio for low-grade glioma associated with rs55705857 was 4.3 (P = 2.31 × 10(-94)). rs55705857 maps to a highly evolutionarily conserved sequence within the long non-coding RNA CCDC26 raising the possibility of direct functionality. These data provide additional insights into the aetiological basis of glioma development.

Interlaboratory comparison of IDH mutation detection.

Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.