The genomic landscape of TERT promoter wildtype-IDH wildtype glioblastoma.

The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTpWT-IDHWT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTpWT-IDHWT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDHWT-TERTSV), and an ALT-positive subgroup (IDHWT-ALT) with mutations in ATRX or SMARCAL1.

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

BACKGROUND: Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. METHODS: TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. RESULTS: TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. CONCLUSIONS: TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets.

Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas.

Gliomas arising in the brainstem and thalamus are devastating tumors that are difficult to surgically resect. To determine the genetic and epigenetic landscape of these tumors, we performed exomic sequencing of 14 brainstem gliomas (BSGs) and 12 thalamic gliomas. We also performed targeted mutational analysis of an additional 24 such tumors and genome-wide methylation profiling of 45 gliomas. This study led to the discovery of tumor-specific mutations in PPM1D, encoding wild-type p53-induced protein phosphatase 1D (WIP1), in 37.5% of the BSGs that harbored hallmark H3F3A mutations encoding p.Lys27Met substitutions. PPM1D mutations were mutually exclusive with TP53 mutations in BSG and attenuated p53 activation in vitro. PPM1D mutations were truncating alterations in exon 6 that enhanced the ability of PPM1D to suppress the activation of the DNA damage response checkpoint protein CHK2. These results define PPM1D as a frequent target of somatic mutation and as a potential therapeutic target in brainstem gliomas.

Mutations in IDH1, IDH2, and in the TERT promoter define clinically distinct subgroups of adult malignant gliomas.

Frequent mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and the promoter of telomerase reverse transcriptase (TERT) represent two significant discoveries in glioma genomics. Understanding the degree to which these two mutations co-occur or occur exclusively of one another in glioma subtypes presents a unique opportunity to guide glioma classification and prognosis. We analyzed the relationship between overall survival (OS) and the presence of IDH1/2 and TERT promoter mutations in a panel of 473 adult gliomas. We hypothesized and show that genetic signatures capable of distinguishing among several types of gliomas could be established providing clinically relevant information that can serve as an adjunct to histopathological diagnosis. We found that mutations in the TERT promoter occurred in 74.2% of glioblastomas (GBM), but occurred in a minority of Grade II-III astrocytomas (18.2%). In contrast, IDH1/2 mutations were observed in 78.4% of Grade II-III astrocytomas, but were uncommon in primary GBM. In oligodendrogliomas, TERT promoter and IDH1/2 mutations co-occurred in 79% of cases. Patients whose Grade III-IV gliomas exhibit TERT promoter mutations alone predominately have primary GBMs associated with poor median OS (11.5 months). Patients whose Grade III-IV gliomas exhibit IDH1/2 mutations alone predominately have astrocytic morphologies and exhibit a median OS of 57 months while patients whose tumors exhibit both TERT promoter and IDH1/2 mutations predominately exhibit oligodendroglial morphologies and exhibit median OS of 125 months. Analyzing gliomas based on their genetic signatures allows for the stratification of these patients into distinct cohorts, with unique prognosis and survival.

The genetic landscape of anaplastic astrocytoma.

Anaplastic astrocytoma WHO grade III (A3) is a lethal brain tumor that often occurs in middle aged patients. Clinically, it is challenging to distinguish A3 from glioblastoma multiforme (GBM) WHO grade IV. To reveal the genetic landscape of this tumor type, we sequenced the exome of a cohort of A3s (n=16). For comparison and to illuminate the genomic landscape of other glioma subtypes, we also included in our study diffuse astrocytoma WHO grade II (A2, n=7), oligoastrocytoma WHO grade II (OA2, n=2), anaplastic oligoastrocytoma WHO grade III (OA3, n=4), and GBM (n=28). Exome sequencing of A3s identified frequent mutations in IDH1 (75%, 12/16), ATRX (63%, 10/16), and TP53 (82%, 13/16). In contrast, the majority of GBMs (75%, 21/28) did not contain IDH1 or ATRX mutations, and displayed a distinct spectrum of mutations. Finally, our study also identified novel genes that were not previously linked to this tumor type. In particular, we found mutations in Notch pathway genes (NOTCH1, NOTCH2, NOTCH4, NOTCH2NL), including a recurrent NOTCH1-A465Tmutation, in 31% (5/16) of A3s. This study suggests genetic signatures will be useful for the classification of gliomas.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas.

Mutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1, which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutations in many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas ( more than 10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.

Integrated genomic analyses identify ERRFI1 and TACC3 as glioblastoma-targeted genes.

The glioblastoma genome displays remarkable chromosomal aberrations, which harbor critical glioblastoma-specific genes contributing to several oncogenetic pathways. To identify glioblastoma-targeted genes, we completed a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in glioblastomas. We performed copy number analysis of 111 glioblastomas by Digital Karyotyping and Illumina BeadChip assays and validated our findings using data from the TCGA (The Cancer Genome Atlas) glioblastoma project. From this study, we identified recurrent focal copy number alterations in 1p36.23 and 4p16.3. Expression analyses of genes located in the two regions revealed genes which are dysregulated in glioblastomas. Specifically, we identify EGFR negative regulator, ERRFI1, within the minimal region of deletion in 1p36.23. In glioblastoma cells with a focal deletion of the ERRFI1 locus, restoration of ERRFI1 expression slowed cell migration. Furthermore, we demonstrate that TACC3, an Aurora-A kinase substrate, on 4p16.3, displays gain of copy number, is overexpressed in a glioma-grade-specific pattern, and correlates with Aurora kinase overexpression in glioblastomas. Our multifaceted genomic evaluation of glioblastoma establishes ERRFI1 as a potential candidate tumor suppressor gene and TACC3 as a potential oncogene, and provides insight on targets for oncogenic pathway-based therapy.