Dataset Information

Sensitive and rapid detection of TERT promoter and IDH mutations in diffuse gliomas.

ABSTRACT: BACKGROUND:Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations. METHODS:We evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples. RESULTS:GliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity. CONCLUSIONS:Our results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.

SUBMITTER: Diplas BH

PROVIDER: S-EPMC6422442 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Sensitive and rapid detection of TERT promoter and IDH mutations in diffuse gliomas.

Diplas Bill H BH Liu Heng H Yang Rui R Hansen Landon J LJ Zachem Alexis L AL Zhao Fangping F Bigner Darell D DD McLendon Roger E RE Jiao Yuchen Y He Yiping Y Waitkus Matthew S MS Yan Hai H

Neuro-oncology 20190301 4

<h4>Background</h4>Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations.<h4> ...[more]

PMID: 30346624

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Project description:IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations (p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup.

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.

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:Gliomas are the most common type of primary brain tumor, yet the prognosis for glioma patients remains poor. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTp) are associated with diagnosis and poor prognosis in gliomas. Here, we developed a precise and rapid Sanger sequencing assay to screen or TERTp mutations. We established the Sanger sequencing approach for the detection of TERTp mutations based on human glioma cell lines U251 and assessed the analytical validation by determining the accuracy, sensitivity, precision, and specificity. In our study, we verified the accuracy of Sanger sequencing by the real-time polymerase chain reaction method. Our data showed that TERTp mutations were detected at an analytical sensitivity of 10% per mutant. The precision and specificity validation also showed the desired results. In total, 147 glioma patients were investigated for TERTp mutations, and of each patient, clinical data and molecular characteristics were analyzed. We found that anaplastic oligodendroglioma had the highest frequency of TERTp mutations (66.7%). No differences in TERTp mutation frequency were observed between frozen tissue specimens and formalin-fixed and paraffin-embedded tissue. TERTp mutations were associated with older patients (≥45 years), whereas isocitrate dehydrogenase (IDH) mutations were inclined to a younger age (<45 years), frontal location, and pathologic stage II-III patients. IDH mutations were significantly associated with O6-methylguanine-DNA methyltransferase (MGMT) methylation (P = 0.003) and lower Ki-67 protein expression (P = 0.011). Moreover, MGMT methylation was enriched in IDH-mutant/TERTp-mutant gliomas, and Ki-67 protein expression was the highest in the IDH-wild type/TERTp-mutant group. Taken together, the findings of this study indicate the establishment of a rapid, precise, and practical Sanger sequencing technique for TERTp mutations in gliomas that may show promising results in clinical applications.

Project description:ObjectivesGliomas with comutations of isocitrate dehydrogenase (IDH) genes and telomerase reverse transcriptase (TERT) gene promoter (IDHmut pTERTmut) show distinct biological features and respond to first-line treatment differently in comparison with other gliomas. This study aimed to characterize the IDHmut pTERTmut gliomas in multimodal MRI using the radiomic method and establish a precise diagnostic model identifying this group of gliomas.MethodsA total of 140 patients with untreated primary gliomas were admitted between 2016 and 2020 to West China Hospital as a discovery cohort, including 22 IDHmut pTERTmut patients. Thirty-four additional cases from a different hospital were included in the study as an independent validation cohort. A total of 3654 radiomic features were extracted from the preoperative multimodal MRI images (T1c, FLAIR, and ADC maps) and filtered in a data-driven approach. The discovery cohort was split into training and test sets by a 4:1 ratio. A diagnostic model (multilayer perceptron classifier) for detecting the IDHmut pTERTmut gliomas was trained using an automatic machine-learning algorithm named tree-based pipeline optimization tool (TPOT). The most critical radiomic features in the model were identified and visualized.ResultsThe model achieved an area under the receiver-operating curve (AUROC) of 0.971 (95% CI, 0.902-1.000), the sensitivity of 0.833 (95% CI, 0.333-1.000), and the specificity of 0.966 (95% CI, 0.931-1.000) in the test set. The area under the precision-recall curve (AUCPR) was 0.754 (95% CI, 0.572-0.833) and the F1 score was 0.833 (95% CI, 0.500-1.000). In the independent validation set, the model reached 0.952 AUROC, 0.714 sensitivity, 0.963 specificity, 0.841 AUCPR, and 0.769 F1 score. MR radiomic features of the IDHmut pTERTmut gliomas represented homogenous low-complexity texture in three modalities.ConclusionsAn accurate diagnostic model was constructed for detecting IDHmut pTERTmut gliomas using multimodal radiomic features. The most important features were associated with the homogenous simple texture of IDHmut pTERTmut gliomas in MRI images transformed using Laplacian of Gaussian and wavelet filters.

Dataset Information

Sensitive and rapid detection of TERT promoter and IDH mutations in diffuse gliomas.

Publications

Sensitive and rapid detection of TERT promoter and IDH mutations in diffuse gliomas.

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