Molecular landscape of IDH-mutant primary astrocytoma Grade IV/glioblastomas.

WHO 2016 classified glioblastomas into IDH-mutant and IDH-wildtype with the former having a better prognosis but there was no study on IDH-mutant primary glioblastomas only, as previous series included secondary glioblastomas. We recruited a series of 67 IDH-mutant primary glioblastomas/astrocytoma IV without a prior low-grade astrocytoma and examined them using DNA-methylation profiling, targeted sequencing, RNA sequencing and TERT promoter sequencing, and correlated the molecular findings with clinical parameters. The median OS of 39.4 months of 64 cases and PFS of 25.9 months of 57 cases were better than the survival data of IDH-wildtype glioblastomas and IDH-mutant secondary glioblastomas retrieved from datasets. The molecular features often seen in glioblastomas, such as EGFR amplification, combined +7/-10, and TERT promoter mutations were only observed in 6/53 (11.3%), 4/53 (7.5%), and 2/67 (3.0%) cases, respectively, and gene fusions were found only in two cases. The main mechanism for telomere maintenance appeared to be alternative lengthening of telomeres as ATRX mutation was found in 34/53 (64.2%) cases. In t-SNE analyses of DNA-methylation profiles, with an exceptional of one case, a majority of our cases clustered to IDH-mutant high-grade astrocytoma subclass (40/53; 75.5%) and the rest to IDH-mutant astrocytoma subclass (12/53; 22.6%). The latter was also enriched with G-CIMP high cases (12/12; 100%). G-CIMP-high status and MGMT promoter methylation were independent good prognosticators for OS (p = 0.022 and p = 0.002, respectively) and TP53 mutation was an independent poor prognosticator (p = 0.013) when correlated with other clinical parameters. Homozygous deletion of CDKN2A/B was not correlated with OS (p = 0.197) and PFS (p = 0.278). PDGFRA amplification or mutation was found in 16/59 (27.1%) of cases and was correlated with G-CIMP-low status (p = 0.010). Aside from the three well-known pathways of pathogenesis in glioblastomas, chromatin modifying and mismatch repair pathways were common aberrations (88.7% and 20.8%, respectively), the former due to high frequency of ATRX involvement. We conclude that IDH-mutant primary glioblastomas have better prognosis than secondary glioblastomas and have major molecular differences from other commoner glioblastomas. G-CIMP subgroups, MGMT promoter methylation, and TP53 mutation are useful prognostic adjuncts.

Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9.

Cancer cells are defined genetically by the mutations they harbor, commonly single nucleotide substitutions. Therapeutic approaches which specifically target cancer cells by recognizing these defining genetic aberrations are expected to exhibit minimal side-effects. However, current protein-based targeted therapy is greatly limited by the range of genes that can be targeted, as well as by acquired resistance. We hypothesized that a therapeutic oligonucleotide-based strategy may address this need of specific cancer targeting. We used CRISPR/Cas9 system to target a commonly occurring EGFR point mutation, L858R, with an oligonucleotide guide that recognizes L858R as the suitable protospacer-adjacent motif (PAM) sequence for DNA cleavage. We found that this strategy, which utilized PAM to differentiate cancer mutation from normal, afforded high specificity to the extent of a single nucleotide substitution. The anti-L858R vehicle resulted in selective genome cleavage only in L858R mutant cells, as detected by Sanger sequencing and T7 Endonuclease I assay. Wild-type cells were unaffected by the same treatment. Digital PCR revealed 37.9 ± 8.57% of L858R gene copies were targeted in mutant. Only treated mutant cells, but not wild-type cells, showed reduction in EGFR expression and decreased cell proliferation. Treated mutant cells also formed smaller tumor load in vivo. This targeting approach is expected to be able to target a significant subset of the 15-35% cancer mutations with C > G, A > G, and T > G point mutations. Thus, this strategy may serve as a useful approach to target cancer-defining mutations with specificity, to the extent of differentiating the change of a single nucleotide.

Pediatric low-grade gliomas can be molecularly stratified for risk.

Pediatric low-grade gliomas (PLGGs) consist of a number of entities with overlapping histological features. PLGGs have much better prognosis than the adult counterparts, but a significant proportion of PLGGs suffers from tumor progression and recurrence. It has been shown that pediatric and adult low-grade gliomas are molecularly distinct. Yet the clinical significance of some of newer biomarkers discovered by genomic studies has not been fully investigated. In this study, we evaluated in a large cohort of 289 PLGGs a list of biomarkers and examined their clinical relevance. TERT promoter (TERTp), H3F3A and BRAF V600E mutations were detected by direct sequencing. ATRX nuclear loss was examined by immunohistochemistry. CDKN2A deletion, KIAA1549-BRAF fusion, and MYB amplification were determined by fluorescence in situ hybridization (FISH). TERTp, H3F3A, and BRAF V600E mutations were identified in 2.5, 6.4, and 7.4% of PLGGs, respectively. ATRX loss was found in 4.9% of PLGGs. CDKN2A deletion, KIAA1549-BRAF fusion and MYB amplification were detected in 8.8, 32.0 and 10.6% of PLGGs, respectively. Survival analysis revealed that TERTp mutation, H3F3A mutation, and ATRX loss were significantly associated with poor PFS (p < 0.0001, p < 0.0001, and p = 0.0002) and OS (p < 0.0001, p < 0.0001, and p < 0.0001). BRAF V600E was associated with shorter PFS (p = 0.011) and OS (p = 0.032) in a subset of PLGGs. KIAA1549-BRAF fusion was a good prognostic marker for longer PFS (p = 0.0017) and OS (p = 0.0029). MYB amplification was also a favorable marker for a longer PFS (p = 0.040). Importantly, we showed that these molecular biomarkers can be used to stratify PLGGs into low- (KIAA1549-BRAF fusion or MYB amplification), intermediate-I (BRAF V600E and/or CDKN2A deletion), intermediate-II (no biomarker), and high-risk (TERTp or H3F3A mutation or ATRX loss) groups with distinct PFS (p < 0.0001) and OS (p < 0.0001). This scheme should aid in clinical decision-making.

Oligodendrogliomas in pediatric and teenage patients only rarely exhibit molecular markers and patients have excellent survivals.

Although oligodendrogliomas appear histologically similar in adult and pediatric patients, the latter have only been rarely studied and most of those studies did not have long follow-up. We examined 55 oligodendroglial tumors from pediatric and teenage patients for their biomarkers with formalin-fixed paraffin-embedded tissues and studied their survival status. None of the tumors harbored 1p/19q codeletion or IDH mutation. Mutations in TERTp (4%), BRAF (11%), FGFR1 (3%) and H3F3A (5%), fusions of BRAF (8%) and FGFR1 (8%) were found sparingly and almost all in a mutually exclusive manner. Molecular events were exclusively found in tumors with classic oligodendroglial histology. Survival analysis showed remarkably excellent prognosis compared to the adult counterparts. 5-year overall survival was 95% in our cohort with median follow-up of 8.1 years and in nine patients with follow-up more than 10 years, the 10-year overall survival was 100%. The 5-year and 10-year progression-free survivals of our cohort were 89 and 77%, respectively. FGFR1 fusion seemed to confer a poor prognosis in pediatric oligodendrogliomas. Patients receiving adjuvant chemotherapy (p = 0.046) or harboring Grade II histology (p < 0.001) had longer interval to recurrence. Our study demonstrated the distinct indolent clinical course of pediatric and teenage oligodendrogliomas compared to the adult tumors. Molecular markers commonly seen in adult oligodendrogliomas and other pediatric low-grade gliomas were only rarely seen. Since there is no clinical or molecular evidence suggesting that pediatric "oligodendrogliomas" are the same as adult oligodendrogliomas albeit histologic similarity, a case can be made for their separation from adult oligodendrogliomas in the next WHO classification.

The kinesin KIF14 is overexpressed in medulloblastoma and downregulation of KIF14 suppressed tumor proliferation and induced apoptosis.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. At present, there is no well-established targeted drug for majority of patients. The kinesin family member 14 (KIF14) is a novel oncogene located on chromosome 1q and is dysregulated in multiple cancers. The objectives of this study were to evaluate KIF14 expression and chromosome 1q copy number in MB, and to delineate its biological functions in MB pathogenesis. By quantitative RT-PCR and immunohistochemistry, we found KIF14 was overexpressed in MB. Increased KIF14 expression at protein level was strongly associated with shorter progression-free survival (P=0.0063) and overall survival (P=0.0083). Fluorescence in situ hybridization (FISH) analysis confirmed genomic gain of chromosome 1q in 17/93 (18.3%) of MB. Combined genetic and immunohistochemical analyses revealed that 76.5% of MB with 1q gain showed consistent overexpression of KIF14, and a tight link between chromosome 1q gain and KIF14 overexpression (P=0.03). Transient, siRNAs-mediated downregulation of KIF14 suppressed cell proliferation and induced apoptosis in two MB cell lines. Stably KIF14 knockdown by shRNAs inhibited cell viability, colony formation, migration and invasion, and tumor sphere formation in MB cells. We conclude that KIF14 is dysregulated in MB and is an adverse prognostic factor for survival. Furthermore, KIF14 is part of MB biology and is a potential therapeutic target for MB.

TERT promoter mutations contribute to subset prognostication of lower-grade gliomas.

Recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) have been found in various cancers including diffuse gliomas. Mutations lead to TERT upregulation and are associated with aggressive clinical behavior in glioblastomas. However, the clinical significance of TERT promoter mutations in lower-grade gliomas remains undetermined. The aim of this study is to evaluate the status of TERT promoter and the respective prognostic significance in a cohort of 237 lower-grade gliomas comprising grades II and III astrocytomas, oligodendrogliomas, and oligoastrocytomas. Mutually exclusive mutations in TERT promoter, C228T and C250T, were identified in 16/105 (15%) diffuse astrocytomas, 16/63 (25%) anaplastic astrocytomas, 13/18 (72%) oligodendrogliomas, 3/3 (100%) anaplastic oligodendrogliomas, 17/45 (38%) oligoastrocytomas, and 2/3 (67%) anaplastic oligoastrocytomas. Mutations co-occurred with 1p/19q codeletion (P<0.001) and are associated with oligodendroglial histology (P<0.001). Kaplan-Meier's survival analysis showed that TERT promoter mutation (P=0.037), Isocitrate dehydrogenase (IDH) mutation (P<0.001), and 1p/19q codeletion (P<0.001) were associated with favorable overall survival (OS). In the subset of 116 IDH-mutated lower-grade gliomas lacking 1p/19q codeletion, 19 TERT promoter-mutated tumors exhibited longer progression-free survival (PFS) (P=0.027) and OS (P=0.004). Consistent with this observation, in the subset of 97 IDH-mutated astrocytomas, 14 TERT promoter-mutated tumors showed longer PFS (P=0.001) and OS (P=0.001). In contrast, among the subset of 74 IDH wild-type lower-grade gliomas with intact 1p/19q, TERT promoter mutation was associated with shorter PFS (P=0.001) and OS (P=0.001). Similarly, in the subset of 65 IDH wild-type astrocytomas, 16 TERT promoter-mutated tumors exhibited unfavorable PFS (P=0.007) and OS (P=0.008). Our results indicate that when combined with IDH status, TERT promoter mutation contributes to prognostic subgroups of lower-grade astrocytic tumors or 1p/19q intact lower-grade gliomas and this may further refine future molecular classification of lower-grade gliomas.

Loss of CIC and FUBP1 expressions are potential markers of shorter time to recurrence in oligodendroglial tumors.

Combined deletion of chromosomes 1p and 19q is a prognostic marker in oligodendroglial tumors. Recent studies in oligodendroglial tumors have unveiled recurrent mutations of CIC (homolog of Drosophila capicua) and FUBP1 (far upstream element binding protein 1) that are located on 19q13 and 1p31, respectively. However, the impact of CIC and FUBP1 mutations on their protein expressions has not been examined. The aims of this study were to correlate the expression patterns of CIC and FUBP1 with their mutation profiles and to evaluate the clinical relevance of these molecular markers in 55 oligodendroglial tumors diagnosed in 47 adult patients. Using direct sequencing, somatic mutations of CIC and FUBP1 were identified in 47% (22/47) and 16% (7/45) of oligodendroglial tumors, respectively. Immunohistochemical analysis revealed loss of CIC or FUBP1 protein expression in 36% (20/55) and 16% (9/55) of oligodendroglial tumors examined. Somatic mutation was significantly associated with absent protein expression for both genes (CIC, P=0.01; FUBP1, P=0.00001). Four tumors with undetectable CIC mutations exhibited absent CIC expression, suggesting that CIC inactivation could be mediated by mechanisms other than mutations and genomic loss. Univariate survival analysis revealed that 1p/19q codeletion was significantly associated with overall survival (P=0.05). Loss of CIC expression was significantly correlated with shorter progression-free survival (P=0.03), whereas CIC alteration (mutation or null expression) with worse overall survival (P=0.05). Absent FUBP1 expression was linked with unfavorable progression-free survival (P=0.02) and overall survival (P=0.01). In 16 tumors with 1p/19q codeletion, CIC mutation was associated with unfavorable survival (P=0.01). There was a correlation between lack of CIC or FUBP1 expression and poor progression-free survival (P=0.004; P=0.0003). No molecular markers showed association with survival in oligodendroglial tumors lacking 1p/19q codeletion. We conclude that absent CIC and FUBP1 expressions are potential markers of shorter time to recurrence and CIC mutation a potential marker of worse prognosis, especially in tumors carrying 1p/19q codeletion.

Identifying predictors of glioma evolution from longitudinal sequencing.

Clonal evolution drives cancer progression and therapeutic resistance. Recent studies have revealed divergent longitudinal trajectories in gliomas, but early molecular features steering posttreatment cancer evolution remain unclear. Here, we collected sequencing and clinical data of initial-recurrent tumor pairs from 544 adult diffuse gliomas and performed multivariate analysis to identify early molecular predictors of tumor evolution in three diffuse glioma subtypes. We found that CDKN2A deletion at initial diagnosis preceded tumor necrosis and microvascular proliferation that occur at later stages of IDH-mutant glioma. Ki67 expression at diagnosis was positively correlated with acquiring hypermutation at recurrence in the IDH-wild-type glioma. In all glioma subtypes, MYC gain or MYC-target activation at diagnosis was associated with treatment-induced hypermutation at recurrence. To predict glioma evolution, we constructed CELLO2 (Cancer EvoLution for LOngitudinal data version 2), a machine learning model integrating features at diagnosis to forecast hypermutation and progression after treatment. CELLO2 successfully stratified patients into subgroups with distinct prognoses and identified a high-risk patient group featured by MYC gain with worse post-progression survival, from the low-grade IDH-mutant-noncodel subtype. We then performed chronic temozolomide-induction experiments in glioma cell lines and isogenic patient-derived gliomaspheres and demonstrated that MYC drives temozolomide resistance by promoting hypermutation. Mechanistically, we demonstrated that, by binding to open chromatin and transcriptionally active genomic regions, c-MYC increases the vulnerability of key mismatch repair genes to treatment-induced mutagenesis, thus triggering hypermutation. This study reveals early predictors of cancer evolution under therapy and provides a resource for precision oncology targeting cancer dynamics in diffuse gliomas.

Combinations of Single-Gene Biomarkers Can Precisely Stratify 1,028 Adult Gliomas for Prognostication.

Advanced genomic techniques have now been incorporated into diagnostic practice in neuro-oncology in the literature. However, these assays are expensive and time-consuming and demand bioinformatics expertise for data interpretation. In contrast, single-gene tests can be run much more cheaply, with a short turnaround time, and are available in general pathology laboratories. The objective of this study was to establish a molecular grading scheme for adult gliomas using combinations of commonly available single-gene tests. We retrospectively evaluated molecular diagnostic data of 1,275 cases of adult diffuse gliomas from three institutions where we were testing for IDH1/2 mutation, TERTp mutation, 1p19q codeletion, EGFR amplification, 10q deletion, BRAF V600E, and H3 mutations liberally in our regular diagnostic workup. We found that a molecular grading scheme of Group 1 (1p19q codeleted, IDH mutant), Group 2 (IDH mutant, 1p19q non-deleted, TERT mutant), Group 3 (IDH mutant, 1p19q non-deleted, TERT wild type), Group 4 (IDH wild type, BRAF mutant), Group 5 (IDH wild type, BRAF wild type and not possessing the criteria of Group 6), and Group 6 (IDH wild type, and any one of TERT mutant, EGFR amplification, 10q deletion, or H3 mutant) could significantly stratify this large cohort of gliomas for risk. A total of 1,028 (80.6%) cases were thus classifiable with sufficient molecular data. There were 270 cases of molecular Group 1, 59 cases of molecular Group 2, 248 cases of molecular Group 3, 27 cases of molecular Group 4, 117 cases of molecular Group 5, and 307 cases of molecular Group 6. The molecular groups were independent prognosticators by multivariate analyses and in specific instances, superseded conventional histological grades. We were also able to validate the usefulness of the Groups with a cohort retrieved from The Cancer Genome Atlas (TCGA) where similar molecular tests were liberally available. We conclude that a single-gene molecular stratification system, useful for fine prognostication, is feasible and can be adopted by a general pathology laboratory.

Establishment and characterization of meningioma patient-derived organoid.

Meningioma is a central nervous system tumor originated from arachnoid cells. 2D cell culture is widely used as a platform for tumor research as it enables us to culture cells in in vitro and a controlled environment. However, in 2D culture condition, 3D architecture of in vivo tumor mass is lost and phenotypic change may occur. Due to the drawbacks of 2D cell culture, organoid culture is seen as an alternative platform for disease modeling, drug testing and personalized medicine. The objective of this study was to establishing protocol for culturing cells from patient meningioma tissue in in vitro 3D environment. Eight meningiomas were collected for the 3D organoid culture. Cells of 5 meningioma tissues survived and proliferated. Under 3D culture condition, cell aggregates were formed and cytoplasmic processes linking the cell aggregates could be observed. In H&E staining, ovaloid cells and spindle cells were observed. Resembling cultured organoids observed under the light microscope, cell aggregates were also observed in the H&E staining. Epithelial Membrane Antigen (EMA) staining was positive. In 4 (80%) cultured organoids, low Ki67 index (≤6%) were measured. In one cultured organoid, a high Ki67 index (12.8%) was seen. The result of this study revealed the feasibility of culturing meningioma cells in in vitro 3D culture condition. Organoid technology showed its potential as an alternative platform for meningioma research.

Mismatch repair proteins PMS2 and MLH1 can further refine molecular stratification of IDH-mutant lower grade astrocytomas.

The diagnostic role of Isocitrate Dehydrogenase (IDH) mutation status in adult lower grade astrocytomas was first formally presented within the WHO Classification of Tumours of the Central Nervous System (2016). IDH-mutant astrocytomas are not as common as IDH-wildtype astrocytomas but are of better prognosis. Our previous study provided an evident that IDH-mutant lower grade astrocytomas is not a homogeneous group and could be further stratified by PDGFRA amplification, CDK4 amplification and CDKN2A deletion. In this study, we detected the expressions of DNA mismatch repair (MMR) proteins (PMS2, MLH1, MSH2, MSH6) and PD-L1 by immunohistochemistry in 147 IDH-mutant lower grade astrocytomas and explored their clinical relevance. The loss of was identified in 28.6%, 1.4%, 8.8% and 13.6%, respectively. PD-L1 expression was detected in 1.4% of this cohort. Survival analysis revealed that loss of PMS2 was correlated with shorter OS (p < 0.001) and PFS (p = 0.005). Loss of PMS2 or MLH1 was associated with shorter OS (p < 0.001) and PFS (p = 0.008). In IDH-mutant lower grade astrocytomas without CDKN2A deletion, loss of PMS2 was associated with poorer OS (p < 0.001) and PFS (p = 0.001). Furthermore, among IDH-mutant lower grade astrocytomas lacking the three biomarkers (PDGFRA, CDK4 and CDKN2A), loss of PMS2 was also associated with a poorer OS (p < 0.001) and PFS (p = 0.003). Our data illustrated the potential application of MMR genes in stratification of IDH-mutant lower grade astrocytomas without PDGFRA, CDK4 and CDKN2A copy number alterations.

Clinical and mutational profiles of adult medulloblastoma groups.

Adult medulloblastomas are clinically and molecularly understudied due to their rarity. We performed molecular grouping, targeted sequencing, and TERT promoter Sanger sequencing on a cohort of 99 adult medulloblastomas. SHH made up 50% of the cohort, whereas Group 3 (13%) was present in comparable proportion to WNT (19%) and Group 4 (18%). In contrast to paediatric medulloblastomas, molecular groups had no prognostic impact in our adult cohort (p = 0.877). Most frequently mutated genes were TERT (including promoter mutations, mutated in 36% cases), chromatin modifiers KMT2D (31%) and KMT2C (30%), TCF4 (31%), PTCH1 (27%) and DDX3X (24%). Adult WNT patients showed enrichment of TP53 mutations (6/15 WNT cases), and 3/6 TP53-mutant WNT tumours were of large cell/anaplastic histology. Adult SHH medulloblastomas had frequent upstream pathway alterations (PTCH1 and SMO mutations) and few downstream alterations (SUFU mutations, MYCN amplifications). TERT promoter mutations were found in 72% of adult SHH patients, and were restricted to this group. Adult Group 3 tumours lacked hallmark MYC amplifications, but had recurrent mutations in KBTBD4 and NOTCH1. Adult Group 4 tumours harboured recurrent mutations in TCF4 and chromatin modifier genes. Overall, amplifications of MYC and MYCN were rare (3%). Since molecular groups were not prognostic, alternative prognostic markers are needed for adult medulloblastoma. KMT2C mutations were frequently found across molecular groups and were associated with poor survival (p = 0.002). Multivariate analysis identified histological type (p = 0.026), metastasis (p = 0.031) and KMT2C mutational status (p = 0.046) as independent prognosticators in our cohort. In summary, we identified distinct clinical and mutational characteristics of adult medulloblastomas that will inform their risk stratification and treatment.

IDH mutant lower grade (WHO Grades II/III) astrocytomas can be stratified for risk by CDKN2A, CDK4 and PDGFRA copy number alterations.

In the 2016, WHO classification of tumors of the central nervous system, isocitrate dehydrogenase (IDH) mutation is a main classifier for lower grade astrocytomas and IDH-mutated astrocytomas is now regarded as a single group with longer survival. However, the molecular and clinical heterogeneity among IDH mutant lower grade (WHO Grades II/III) astrocytomas have only rarely been investigated. In this study, we recruited 160 IDH mutant lower grade (WHO Grades II/III) astrocytomas, and examined PDGFRA amplification, CDKN2A deletion and CDK4 amplification by FISH analysis, TERT promoter mutation by Sanger sequencing and ATRX loss and p53 expression by immunohistochemistry. We identified PDGFRA amplification, CDKN2A homozygous deletion and CDK4 amplification in 18.8%, 15.0% and 18.1% of our cohort respectively, and these alterations occurred in a mutually exclusive fashion. PDGFRA amplification was associated with shorter PFS (P = 0.0003) and OS (P < 0.0001). In tumors without PDGFRA amplification, CDKN2A homozygous deletion or CDK4 amplification was associated with a shorter OS (P = 0.035). Tumors were divided into three risk groups based on the presence of molecular alterations: high risk (PDGFRA amplification), intermediate risk (CDKN2A deletion or CDK4 amplification) and low risk (neither CDKN2A deletion and CDK4 amplification nor PDGFRA amplification). These three risk groups were significantly different in overall survival with mean survivals of 40.5, 62.9 and 71.5 months. The high-risk group also demonstrated a shorter PFS compared to intermediate- (P = 0.036) and low-risk (P < 0.0001) groups. One limitation of this study is the relatively short follow-up period, a common confounding factor for studies on low-grade tumors. Our data illustrate that IDH mutant lower grade astrocytomas is not a homogeneous group and should be molecularly stratified for risk.

Identification of subsets of IDH-mutant glioblastomas with distinct epigenetic and copy number alterations and stratified clinical risks.

BACKGROUND: IDH-mutant glioblastoma is classified by the 2016 CNS WHO as a group with good prognosis. However, the actual number of cases examined in the literature is relatively small. We hypothesize that IDH-mutant glioblastoma is not a uniform group and should be further stratified. METHODS: We conducted methylation profiles and estimated copy number variations of 57 IDH-mutant glioblastomas. RESULTS: Our results showed that 59.6% and 40.4% of tumors belonged to glioma-CpG island methylator phenotype (G-CIMP)-high and G-CIMP-low methylation subgroups, respectively. G-CIMP-low subgroup was associated with significantly worse overall survival (OS) as compared to G-CIMP-high (P = .005). CDKN2A deletion (42.1%) was the most common gene copy number variation, and was significantly associated with G-CIMP-low subgroup (P = .004). Other frequent copy number changes included mesenchymal-epithelial transition (MET) (5.3%), CCND2 (19.3%), PDGFRA (14.0%), CDK4 (12.3%), and EGFR (12.3%) amplification. Both CDKN2A deletion (P = .036) and MET amplification (P < .001) were associated with poor OS in IDH-mutant glioblastomas. Combined epigenetic signature and gene copy number variations separated IDH-mutant glioblastomas into Group 1 (G-CIMP-high), Group 2 (G-CIMP-low without CDKN2A nor MET alteration), and Group 3 (G-CIMP-low with CDKN2A and/or MET alteration). Survival analysis revealed Groups 1 and 2 exhibited a favorable OS (median survival: 619 d [20.6 mo] and 655 d [21.8 mo], respectively). Group 3 exhibited a significant shorter OS (median survival: 252 d [8.4 mo]). Multivariable analysis confirmed the independent prognostic significance of our Groups. CONCLUSIONS: IDH-mutant glioblastomas should be stratified for risk with combined epigenetic signature and CDKN2A/MET status and some cases have poor outcome.

TERT promoter mutation and its interaction with IDH mutations in glioma: Combined TERT promoter and IDH mutations stratifies lower-grade glioma into distinct survival subgroups-A meta-analysis of aggregate data.

The clinical significance of telomerase reverse transcriptase (TERT) promoter mutation in glioma remains unclear. The aim of our meta-analysis is to investigate the prognostic impact TERT promoter mutation in glioma patients and its interaction with other molecular markers, particularly Isocitrate Dehydrogenase (IDH) mutation from aggregate level data. Relevant articles were searched in four electronic databases including PubMed, Scopus, Web of Science and Virtual Health Library. Pooled HRs were calculated using random effect model weighted by inverse variance method. From 1010 studies, we finally included 28 studies with 11519 patients for meta-analyses. TERT mutation is significantly associated with compromised overall survival (OS) (HR=1.38; 95% CI=1.15-1.67) and progression-free survival (PFS) (HR=1.31; 95% CI=1.06-1.63) in glioma patients. In studying its reaction with IDH, TERT promoter mutation was associated with reduced OS in both IDH-mutant (IDH-mut) and IDH-wild type (IDH-wt) glioblastomas but shown to have inverse effects on IDH-mut and IDH-wt grade II/III tumors. Our analysis categorized WHO grade II/III glioma patients into four distinct survival subgroups with descending survival as follow: TERT-mut/IDH-mut≫TERT-wt/IDH-mut≫TERT-wt/IDH-wt≫TERT-mut/IDH-wt. Prognostic value of TERT promoter mutations in gliomas is dependent on tumor grade and the IDH mutational status. With the same tumor grade in WHO grade II and III tumors and the same IDH mutation status, TERT-mut is a prognostic factor.

Adult IDH wild-type lower-grade gliomas should be further stratified.

BACKGROUND: Astrocytoma of the isocitrate dehydrogenase (IDH) wild-type gene is described as a provisional entity within the new World Health Organization (WHO) classification. Some groups believe that IDH wild-type lower-grade gliomas, when interrogated for other biomarkers, will mostly turn out to be glioblastoma. We hypothesize that not all IDH wild-type lower-grade gliomas have very poor outcomes and the group could be substratified prognostically. METHODS: Seven hundred and eighteen adult WHO grades II and III patients with gliomas from our hospitals were re-reviewed and tested for IDH1/2 mutations. One hundred and sixty-six patients with IDH wild-type cases were identified for further studies, and EGFR and MYB amplifications, mutations of histone H3F3A, TERT promoter (TERTp), and BRAF were examined. RESULTS: EGFR amplification, BRAF, and H3F3A mutations were observed in 13.8%, 6.9%, and 9.5% of patients, respectively, in a mutually exclusive pattern in IDH wild-type lower-grade gliomas. TERTp mutations were detected in 26.8% of cases. Favorable outcome was observed in patients with young age, oligodendroglial phenotype, and grade II histology. Independent adverse prognostic values of older age, nontotal resection, grade III histology, EGFR amplification, and H3F3A mutation were confirmed by multivariable analysis. Tumors were further classified into "molecularly" high grade (harboring EGFR, H3F3A, or TERTp) (median overall survival = 1.23 y) and lower grade (lacking all of the 3) (median overall survival = 7.63 y) with independent prognostic relevance. The most favorable survival was noted in molecularly lower-grade gliomas with MYB amplification. CONCLUSION: Adult IDH wild-type lower-grade gliomas are prognostically heterogeneous and do not have uniformly poor prognosis. Clinical information and additional markers, including MYB, EGFR, TERTp, and H3F3A, should be examined to delineate discrete favorable and unfavorable prognostic groups.

Biomarker-based prognostic stratification of young adult glioblastoma.

While the predominant elderly and the pediatric glioblastomas have been extensively investigated, young adult glioblastomas were understudied. In this study, we sought to stratify young adult glioblastomas by BRAF, H3F3A and IDH1 mutations and examine the clinical relevance of the biomarkers. In 107 glioblastomas aged from 17 to 35 years, mutually exclusive BRAF-V600E (15%), H3F3A-K27M (15.9%), H3F3A-G34R/V (2.8%) and IDH1-R132H (16.8%) mutations were identified in over half of the cases. EGFR amplification and TERTp mutation were only detected in 3.7% and 8.4% in young adult glioblastomas, respectively. BRAF-V600E identified a clinically favorable subset of glioblastomas with younger age, frequent CDKN2A homozygous deletion, and was more amendable to surgical resection. H3F3A-K27M mutated glioblastomas were tightly associated with midline locations and showed dismal prognosis. IDH1-R132H was associated with older age and favorable outcome. Interestingly, tumors with positive PDGFRA immunohistochemical expression exhibited poorer prognosis and identified an aggressive subset of tumors among K27M mutated glioblastomas. Combining BRAF, H3F3A and IDH1 mutations allowed stratification of young adult glioblastomas into four prognostic subgroups. In summary, our study demonstrates the clinical values of stratifying young adult glioblastomas with BRAF, H3F3A and IDH1 mutations, which has important implications in refining prognostic classification of glioblastomas.

Not all 1p/19q non-codeleted oligodendroglial tumors are astrocytic.

Although 1p/19q codeletion is the genetic hallmark defining oligodendrogliomas, approximately 30-40% of oligodendroglial tumors have intact 1p/19q in the literature and they demonstrate a worse prognosis. This group of 1p/19q intact oligodendroglial tumors is frequently suggested to be astrocytic in nature with TP53 and ATRX mutations but actually remains under-investigated. In the present study, we provided evidence that not all 1p/19q intact oligodendroglial tumors are astrocytic through histologic and molecular approaches. We examined 1p/19q status by FISH in a large cohort of 337 oligodendroglial tumors and identified 39.8% lacking 1p/19q codeletion which was independently associated with poor prognosis. Among this 1p/19q intact oligodendroglial tumor cohort, 58 cases demonstrated classic oligodendroglial histology which showed older patient age, better prognosis, association with grade III histology, PDGFRA expression, TERTp mutation, as well as frequent IDH mutation. More than half of the 1p/19q intact oligodendroglial tumors showed lack of astrocytic defining markers, p53 expression and ATRX loss. TP53 mutational analysis was additionally conducted in 45 cases of the 1p/19q intact oligodendroglial tumors. Wild-type TP53 was detected in 71.1% of cases which was associated with classic oligodendroglial histology. Importantly, IDH and TERTp co-occurred in 75% of 1p/19q intact, TP53 wild-type oligodendrogliomas, highlighting the potential of the co-mutations in assisting diagnosis of oligodendrogliomas in tumors with clear cell morphology and non-codeleted 1p/19q status. In summary, our study demonstrated that not all 1p/19q intact oligodendroglial tumors are astrocytic and co-evaluation of IDH and TERTp mutation could potentially serve as an adjunct for diagnosing 1p/19q intact oligodendrogliomas.

TERT promoter mutated WHO grades II and III gliomas are located preferentially in the frontal lobe and avoid the midline.

The promoter region of telomerase reverse transcriptase (TERTp) and isocitrate dehydrogenase (IDH) have been regarded as biomarkers with distinct clinical and phenotypic features. Investigated the possible correlations between tumor location and genetic alterations would enhance our understanding of gliomagenesis and heterogeneity of glioma. We examined mutations of TERTp and IDH by direct sequencing and fluorescence in-situ hybridization in a cohort of 225 grades II and III diffuse gliomas. Correlation analysis between molecular markers and tumor locations was performed by Chi-square tests/Fisher's exact test and multivariate logistic regression analysis. We found gliomas in frontal lobe showed higher frequency of TERTp mutation (P=0.0337) and simultaneously mutations of IDH and TERTp (IDH (mut)-TERTp(mut)) (P=0.0281) than frequency of biomarkers mutation of tumors in no-Frontal lobes, while lower frequency of TERTp mutation (P<0.0001) and simultaneously wild type of IDH and TERTp (IDH (wt)-TERTp(wt)) (P<0.0001) in midline than no-midline lobes. Logistic regression analysis indicated that locations of tumors associated with TERTp mutation (OR=0.540, 95% CI 0.324-0.900, P=0.018) and status of combinations of IDH and TERTp (IDH (mut)-TERTp (mut) vs. IDH (wt)-TERTp (wt) OR=0.162, 95% CI 0.075-0.350, P<0.001). In conclusion, grades II and III gliomas harboring TERTp mutation were located preferentially in the frontal lobe and rarely in midline. Association of IDH-TERTp status and tumor location suggests their potential values in molecular classification of grades II and III gliomas.