A tailored next-generation sequencing panel identified distinct subtypes of wildtype IDH and TERT promoter glioblastomas.
ABSTRACT: Central nervous system tumors are classified based on an integrated diagnosis combining histology and molecular characteristics, including IDH1/2 and H3-K27M mutations, as well as 1p/19q codeletion. Here, we aimed to develop and assess the feasibility of a glioma-tailored 48-gene next-generation sequencing (NGS) panel for integrated glioma diagnosis. We designed a glioma-tailored 48-gene NGS panel for detecting 1p/19q codeletion and mutations in IDH1/2, TP53, PTEN, PDGFRA, NF1, RB1, CDKN2A/B, CDK4, and the TERT promoter (TERTp). We analyzed 106 glioma patients (grade II: 19 cases, grade III: 23 cases, grade IV: 64 cases) using this system. The 1p/19q codeletion was detected precisely in oligodendroglial tumors using our NGS panel. In a cohort of 64 grade ? gliomas, we identified 56 IDH-wildtype glioblastomas. Within these IDH-wildtype glioblastomas, 33 samples (58.9%) showed a mutation in TERTp. Notably, PDGFRA mutations and their amplification were more commonly seen in TERTp-wildtype glioblastomas (43%) than in TERTp-mutant glioblastomas (6%) (P = .001). Hierarchical molecular classification of IDH-wildtype glioblastomas revealed 3 distinct groups of IDH-wildtype glioblastomas. One major cluster was characterized by mutations in PDGFRA, amplification of CDK4 and PDGFRA, homozygous deletion of CDKN2A/B, and absence of TERTp mutations. This cluster was significantly associated with older age (P = .021), higher Ki-67 score (P = .007), poor prognosis (P = .012), and a periventricular tumor location. We report the development of a glioma-tailored NGS panel for detecting 1p/19q codeletion and driver gene mutations on a single platform. Our panel identified distinct subtypes of IDH- and TERTp-wildtype glioblastomas with frequent PDGFRA alterations.
Project description:The "integrated diagnosis" for infiltrating gliomas in the 2016 revised World Health Organization (WHO) classification of tumors of the central nervous system requires assessment of the tumor for IDH mutations and 1p/19q codeletion. Since TERT promoter mutations and ATRX alterations have been shown to be associated with prognosis, we analyzed whether these tumor markers provide additional prognostic information within each of the five WHO 2016 categories. We used data for 1206 patients from the UCSF Adult Glioma Study, the Mayo Clinic and The Cancer Genome Atlas (TCGA) with infiltrative glioma, grades II-IV for whom tumor status for IDH, 1p/19q codeletion, ATRX, and TERT had been determined. All cases were assigned to one of 5 groups following the WHO 2016 diagnostic criteria based on their morphologic features, and IDH and 1p/19q codeletion status. These groups are: (1) Oligodendroglioma, IDH-mutant and 1p/19q-codeleted; (2) Astrocytoma, IDH-mutant; (3) Glioblastoma, IDH-mutant; (4) Glioblastoma, IDH-wildtype; and (5) Astrocytoma, IDH-wildtype. Within each group, we used univariate and multivariate Cox proportional hazards models to assess associations of overall survival with patient age at diagnosis, grade, and ATRX alteration status and/or TERT promoter mutation status. Among Group 1 IDH-mutant 1p/19q-codeleted oligodendrogliomas, the TERT-WT group had significantly worse overall survival than the TERT-MUT group (HR: 2.72, 95% CI 1.05-7.04, p = 0.04). In both Group 2, IDH-mutant astrocytomas and Group 3, IDH-mutant glioblastomas, neither TERT mutations nor ATRX alterations were significantly associated with survival. Among Group 4, IDH-wildtype glioblastomas, ATRX alterations were associated with favorable outcomes (HR: 0.36, 95% CI 0.17-0.81, p = 0.01). Among Group 5, IDH-wildtype astrocytomas, the TERT-WT group had significantly better overall survival than the TERT-MUT group (HR: 0.48, 95% CI 0.27-0.87), p = 0.02). Thus, we present evidence that in certain WHO 2016 diagnostic groups, testing for TERT promoter mutations or ATRX alterations may provide additional useful prognostic information.
Project description: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.
Project description:The World Health Organization (WHO) classification of central nervous system (CNS) tumors was revised in 2016 with a basis on the integrated diagnosis of molecular genetics. We herein provide the guidelines for using molecular genetic tests in routine pathological practice for an accurate diagnosis and appropriate management. While astrocytomas and IDH-mutant (secondary) glioblastomas are characterized by the mutational status of IDH, TP53 , and ATRX , oligodendrogliomas have a 1p/19q codeletion and mutations in IDH, CIC , FUBP1 , and the promoter region of telomerase reverse transcriptase ( TERTp ). IDH-wildtype (primary) glioblastomas typically lack mutations in IDH, but are characterized by copy number variations of EGFR, PTEN, CDKN2A/B, PDGFRA , and NF1 as well as mutations of TERTp . High-grade pediatric gliomas differ from those of adult gliomas, consisting of mutations in H3F3A, ATRX , and DAXX , but not in IDH genes. In contrast, well-circumscribed low-grade neuroepithelial tumors in children, such as pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and ganglioglioma, often have mutations or activating rearrangements in the BRAF, FGFR1, and MYB genes. Other CNS tumors, such as ependymomas, neuronal and glioneuronal tumors, embryonal tumors, meningothelial, and other mesenchymal tumors have important genetic alterations, many of which are diagnostic, prognostic, and predictive markers and therapeutic targets. Therefore, the neuropathological evaluation of brain tumors is increasingly dependent on molecular genetic tests for proper classification, prediction of biological behavior and patient management. Identifying these gene abnormalities requires cost-effective and high-throughput testing, such as next-generation sequencing. Overall, this paper reviews the global guidelines and diagnostic algorithms for molecular genetic testing of brain tumors.
Project description:Background: The latest WHO classification of CNS tumors using the integrated phenotypic and molecular parameters (IDH, ATRX, 1p19q, TERT etc.) have reestablished the CNS tumors classification in addition to traditional histology. The establishment of glioma molecular typing can more accurately predict prognosis, better guide individualized treatment to improve survival. Methods: The expression of IDH1, ATRX, PHH3, P53 and Ki67 was detected by IHC. Molecular status of IDH1/2 and TERT were analyzed using Sanger sequencing. MGMT was explored using methylation-specific PCR. 1p/19q codeletion status was firstly detected by FISH, then further confirmed by multiplex PCR-based next generation sequencing. Results: The mutation frequency of IDH1 was 68.7% (79/115) in WHO II astrocytoma, and 82 cases (82/344, 23.8%) were "triple-negative glioma" in our cohort. Multivariate COX analysis revealed that only IDH, 1p/19q, TERT and MGMT were independent prognostic factors. Noteworthily, we found 7 cases of the new molecular phenotype presented as "IDH wildtype and 1p/19q codeletion", not mentioned in the latest WHO guideline. Conclusion: We detected the newly recommended markers in a large cohort of Chinese glioma patients. Our data demonstrated a relatively lower frequency of IDH mutations and a higher prevalence of triple-negative glioma in Chinese compared with American and European, indicating ethnic and geographical difference in some markers. In addition, the new molecular phenotype "IDH wildtype and 1p/19q codeletion" glioma deserved special focus. These findings suggest that further stratification of infiltrating gliomas is needed for different treatment strategy and precision medicine.
Project description:Background:Oligodendroglioma is genetically defined by concomitant IDH (IDH1/IDH2) mutation and whole-arm 1p/19q codeletion. Codeletion of 1p/19q traditionally evaluated by fluorescence in situ hybridization (FISH) cannot distinguish partial from whole-arm 1p/19q codeletion. Partial 1p/19q codeletion called positive by FISH is diagnostically a "false-positive" result. Chromosomal microarray (CMA) discriminates partial from whole-arm 1p/19q codeletion. Herein, we aimed to estimate the frequency of partial 1p/19q codeletion that would lead to a false-positive FISH result. Methods:FISH 1p/19q codeletion test probe coordinates were mapped onto Oncoscan CMA data to determine the rate of partial 1p/19q codeletion predicted to be positive by FISH. Diffuse astrocytic gliomas with available CMA data (2015-2018) were evaluated and classified based on IDH1-R132H/ATRX/p53 immunohistochemistry, IDH/TERT promoter targeted sequencing, and/or CMA according to classification updates. Predicted false-positive cases were verified by FISH whenever possible. Results:The overall estimated false-positive FISH 1p/19q codeletion rate was 3.6% (8/223). Predicted false positives were verified by FISH in 6 (of 8) cases. False-positive rates did not differ significantly (P = .49) between IDH-mutant (4.6%; 4/86) and IDH-wildtype (2.9%; 4/137) tumors. IDH-wildtype false positives were all WHO grade IV, whereas IDH-mutant false positives spanned WHO grades II-IV. Testing for 1p/19q codeletion would not have been indicated for most false positives based on current classification recommendations. Conclusion:Selective 1p/19q codeletion testing and cautious interpretation for conflicting FISH and histopathological findings are recommended to avoid potential misdiagnosis.
Project description:AbstractBackgroundIsocitrate dehydrogenase (IDH) mutation and 1p/19q-codeletion are oncogenetic alterations with a positive prognostic value for diffuse gliomas, especially grade II and III. Some studies have suggested differences in biological behavior as reflected by radiological characteristics. In this paper, the literature regarding radiological characteristics in grade II and III glioma subtypes was systematically evaluated and a meta-analysis was performed.MethodsStudies that addressed the relationship between conventional radiological characteristics and IDH mutations and/or 1p/19q-codeletions in newly diagnosed, grade II and III gliomas of adult patients were included. The “3-group analysis” compared radiological characteristics between the WHO 2016 glioma subtypes (IDH-mutant astrocytoma, IDH-wildtype astrocytoma, and oligodendroglioma), and the “2-group analysis” compared radiological characteristics between 1p/19q-codeleted gliomas and 1p/19q-intact gliomas.ResultsFourteen studies (3-group analysis: 670 cases, 2-group analysis: 1042 cases) were included. IDH-mutated astrocytomas showed more often sharp borders and less frequently contrast enhancement compared to IDH-wildtype astrocytomas. 1p/19q-codeleted gliomas had less frequently sharp borders, but showed a heterogeneous aspect, calcification, cysts, and edema more frequently. For the 1p/19q-codeleted gliomas, a sensitivity of 96% was found for heterogeneity and a specificity of 88.1% for calcification.ConclusionsSignificant differences in conventional radiological characteristics exist between the WHO 2016 glioma subtypes, which may reflect differences in biological behavior. However, the diagnostic value of the independent radiological characteristics is insufficient to reliably predict the molecular genetic subtype.
Project description:TERT promoter mutations are commonly associated with 1p/19q codeletion in IDH-mutated gliomas. However, whether these mutations have an impact on patient survival independent of 1p/19q codeletion is unknown. In this study, we investigated the impact of TERT promoter mutations on survival in IDH-mutated glioma cases. Detailed clinical information and molecular status data were collected for a cohort of 560 adult patients with IDH-mutated gliomas. Among these patients, 279 had both TERT promoter mutation and 1p/19q codeletion, while 30 had either TERT promoter mutation (n?=?24) or 1p/19q codeletion (n?=?6) alone. A univariable Cox proportional hazard analysis for survival using clinical and genetic factors indicated that a Karnofsky performance status score (KPS) of 90 or 100, WHO grade II or III, TERT promoter mutation, 1p/19q codeletion, radiation therapy, and extent of resection (90-100%) were associated with favorable prognosis (p?<?0.05). A multivariable Cox regression model revealed that TERT promoter mutation had a significantly favorable prognostic impact (hazard ratio?=?0.421, p?=?0.049), while 1p/19q codeletion did not have a significant impact (hazard ratio?=?0.648, p?=?0.349). Analyses incorporating patient clinical and genetic information were further conducted to identify subgroups showing the favorable prognostic impact of TERT promoter mutation. Among the grade II-III glioma patients with a KPS score of 90 or 100, those with IDH-TERT co-mutation and intact 1p/19q (n?=?17) showed significantly longer survival than those with IDH mutation, wild-type TERT, and intact 1p/19q (n?=?185) (5-year overall survival, 94% and 77%, respectively; p?=?0.032). Our results demonstrate that TERT promoter mutation predicts favorable prognosis independent of 1p/19q codeletion in IDH-mutated gliomas. Combined with its adverse effect on survival among IDH-wild glioma cases, the bivalent prognostic impact of TERT promoter mutation may help further refine the molecular diagnosis and prognostication of diffuse gliomas.
Project description:IDH-mutant gliomas are classified into astrocytic or oligodendroglial tumors by 1p/19q status in the WHO 2016 classification, with the latter presenting with characteristic morphology and better prognosis in general. However, the morphological and genetic features within each category are varied, and there might be distinguishable subtypes. We analyzed 170 WHO grade II-IV gliomas resected in our institution. 1p/19q status was analyzed by microsatellite analysis, and genetic mutations were analyzed by next-generation sequencing and Sanger sequencing. For validation, the Brain Lower Grade Glioma dataset of The Cancer Genome Atlas was analyzed. Of the 42 grade III IDH-mutated gliomas, 12 were 1p-intact/19q-intact (anaplastic astrocytomas [AA]), 7 were 1p-intact/19q-loss (AA), and 23 showed 1p/19q-codeletion (anaplastic oligodendrogliomas). Of the 88 IDH-wild type glioblastomas (GBMs), 14 showed 1p-intact/19q-loss status. All of the seven 1p-intact/19q-loss AAs harbored TP53 mutation, but no TERT promotor mutation. All 19q-loss AAs had regions presenting oligodendroglioma-like morphology, and were associated with significantly longer overall survival compared to 19q-intact AAs (P = .001). This tendency was observed in The Cancer Genome Atlas Lower Grade Glioma dataset. In contrast, there was no difference in overall survival between the 19q-loss GBM and 19q-intact GBM (P = .4). In a case of 19q-loss AA, both oligodendroglial morphology and 19q-loss disappeared after recurrence, possibly indicating correlation between 19q-loss and oligodendroglial morphology. We showed that there was a subgroup, although small, of IDH-mutated astrocytomas harboring 19q-loss that present oligodendroglial morphology, and also were associated with significantly better prognosis compared to other 19q-intact astrocytomas.
Project description:BACKGROUND:Accurate classification of diffuse gliomas, the most common tumors of the central nervous system in adults, is important for appropriate treatment. However, detection of isocitrate dehydrogenase (IDH) mutation and chromosome1p/19q codeletion, biomarkers to classify gliomas, is time- and cost-intensive and diagnostic discordance remains an issue. Adenosine to inosine (A-to-I) RNA editing has emerged as a novel cancer prognostic marker, but its value for glioma classification remains largely unexplored. We aim to (1) unravel the relationship between RNA editing and IDH mutation and 1p/19q codeletion and (2) predict IDH mutation and 1p/19q codeletion status using machine learning algorithms. RESULTS:By characterizing genome-wide A-to-I RNA editing signatures of 638 gliomas, we found that tumors without IDH mutation exhibited higher total editing level compared with those carrying it (Kolmogorov-Smirnov test, p?<?0.0001). When tumor grade was considered, however, only grade IV tumors without IDH mutation exhibited higher total editing level. According to 10-fold cross-validation, support vector machines (SVM) outperformed random forest and AdaBoost (DeLong test, p?<?0.05). The area under the receiver operating characteristic curve (AUC) of SVM in predicting IDH mutation and 1p/19q codeletion were 0.989 and 0.990, respectively. After performing feature selection, AUCs of SVM and AdaBoost in predicting IDH mutation were higher than that of random forest (0.985 and 0.983 vs. 0.977; DeLong test, p?<?0.05), but AUCs of the three algorithms in predicting 1p/19q codeletion were similar (0.976-0.982). Furthermore, 67% of the six continuously misclassified samples by our 1p/19q codeletion prediction models were misclassifications in the original labelling after inspection of 1p/19q status and/or pathology report, highlighting the accuracy and clinical utility of our models. CONCLUSIONS:The study represents the first genome-wide analysis of glioma editome and identifies RNA editing as a novel prognostic biomarker for glioma. Our prediction models provide standardized, accurate, reproducible and objective classification of gliomas. Our models are not only useful in clinical decision-making, but also able to identify editing events that have the potential to serve as biomarkers and therapeutic targets in glioma management and treatment.
Project description:Mutations in the telomerase reverse transcriptase gene promoter (TERTp) are common in glioblastomas (GBMs) and oligodendrogliomas (ODGs), and therefore, have a key role in tumorigenesis and may be of prognostic value. However, the extent of their prognostic importance in various gliomas is controversial. We studied 168 patients separated into five groups: Group 1: 65 patients with ODG carrying an IDH1 or IDH2 mutation (IDH-mutant) and 1p/19q-codeletion, Group 2: 23 patients with anaplastic astrocytoma (AA), IDH-mutant, Group 3: 13 patients with GBM, IDH-mutant, Group 4: 15 patients with AA, IDH-wildtype (WT), and Group 5: 52 patients with GBM, IDH-WT. TERTp mutations were found in 96.9%, 4.4%, 76.9%, 20.0%, and 84.6% of patients in Groups 1, 2, 3, 4, and 5, respectively. The R132H mutation in IDH1 was found in 60.5% (23/38) of patients in the AA cohort (Groups 2 and 4) and 20.0% (13/65) of patients from our GBM cohort (Groups 3 and 5), whereas all patients with ODG (Group 1) had a mutation either in IDH1 (n?=?62) or IDH2 (n?=?3). Using Kaplan Meier survival analysis, we found that the TERTp mutation was correlated with poor overall survival (OS) in Groups 2 and 4 combined (P?=?0.001) and in Group 4 (P?=?0.113), and in multivariate analysis, the TERTp mutant group was associated with significantly poor survival in Group 5 (P?=?0.045). However, IDH mutation, MGMT methylation, and younger patient age (<55 years old) were significantly correlated with favorable OS (all P?<?0.05) in our cohort of astrocytic and ODGs. In patients with ODG (Group 1), mutant IDH and TERTp did not have prognostic value because these mutations were universally present. Based on the revised 2016 WHO classification of gliomas, we found that TERTp mutation was frequently present in patients with GBM or ODG and because it was strongly correlated with poor survival outcome in patients with IDH-WT GBM in multivariate analysis, it may be of prognostic value in this subgroup of patients with gliomas.