Classification based on mutations of TERT promoter and IDH characterizes subtypes in grade II/III gliomas

Neuro-Oncology Neuro-Oncology 18(8), 1099 1108, 2016 doi:10.1093/neuonc/now021 Advance Access date 7 March 2016 Classification based on mutations of TERT promoter and IDH characterizes subtypes in grade II/III gliomas Pei Yang, Jinquan Cai, Wei Yan, Wei Zhang, Yinyan Wang, Baoshi Chen, Guilin Li, Shouwei Li, Chenxing Wu, Kun Yao, Wenbin Li, Xiaoxia Peng, Yongping You, Ling Chen, Chuanlu Jiang, Xiaoguang Qiu, and Tao Jiang on behalf of the CGGA project Beijing Neurosurgical Institute, Capital Medical University, Beijing, China (P.Y., W.Z., Y.W., X.Q., T.J.); Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China (P.Y., W.Z., Y.W., B.C., T.J.); Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, China (J.C., C.J.); Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China (W.Y., Y.Y.); Department of Pathology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China (G.L.); Department of Neurosurgery, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing, China (S.L., C.W.); Department of Pathology, Beijing Sanbo Brain Hospital, Capital Medical University, Beijing, China (K.Y.); Department of Oncology, Beijing Shijitan Hospital, Capital Medical University, Beijing, China (W.L.); Department of Epidemiology and Biostatistics, School of Public Health and Family Medicine, Capital Medical University (X.P.); Department of Neurosurgery, Chinese PLA General Hospital, Beijing, China (L.C.); Department of Radiation Therapy, Beijing Tiantan Hospital, Capital Medical University, Beijing, China (X.Q.); China National Clinical Research Center for Neurological Diseases (T.J.) Corresponding Authors: Tao Jiang, MD, PhD, Department of Neuropathology, Beijing Neurosurgical Institute, Capital Medical University; Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University; No. 6 Tiantan Xili, Dongcheng District, Beijing 100050, China (taojiang1964@163.com); Xiaoguang Qiu, Department of Radiation Therapy, Beijing Tiantan Hospital, Capital Medical University; No. 6 Tiantan Xili, Dongcheng District, Beijing 100050, China (ttyy6611@126.com). These authors made equal contributions to this work. Background. Grade II and III gliomas have variable clinical behaviors, showing the distinct molecular genetic alterations from glioblastoma (GBM), many of which eventually transform into more aggressive tumors. Since the classifications of grade II/III gliomas based on the genetic alterations have been recently emerging, it is now a trend to include molecular data into the standard diagnostic algorithm of glioma. Methods. Here we sequenced TERT promoter mutational status (TERTp-mut) in the DNA of 377 grade II/III gliomas and analyzed the clinical factors, molecular aberrations, and transcriptome profiles. Results. We found that TERTp-mut occurred in 145 of 377 grade II and III gliomas (38.5%), mutually exclusive with a TP53 mutation (TP53-mut; P,.001) and coincident with a 1p/19q co-deletion (P ¼.002). TERTp-mut was an independent predictive factor of a good prognosis in all patients (P ¼.048). It has been an independent factor associated with a good outcome in the IDH mutation (IDH-mut) subgroup (P ¼.018), but it has also been associated with a poor outcome in the IDH wild-type (IDH-wt) subgroup (P ¼.049). Combining TERTp-mut and IDH-mut allowed the grade II/III malignancies to be reclassified into IDH-mut/ TERTp-mut, IDH-mut only, TERTp-mut only, and IDH-wt/TERTp-wt. 1p/19q co-deletion, TP53-muts, Ki-67 expression differences, and p-met expression differences characterized IDH-mut/TERTp-mut, IDH-mut only, TERTp-mut only, and IDH-wt/TERTp-wt subtypes, respectively. Conclusions. Our results showed that TERTp-mut combined with IDH-mut allowed simple classification of grade II/III gliomas for stratifying patients and clarifying diagnostic accuracy by supplementing standard histopathological criteria. Keywords: grade II/III gliomas, IDH mutation, TERT promoter mutation, The Cancer Genome Atlas, whole transcriptome sequencing. Received 4 June 2015; accepted 23 January 2016 # The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. 1099

Gliomas are the most common primary malignant tumors of the central nervous system. 1 The conventional tumor grading system is based on histopathological and clinical criteria established by the World Health Organization (WHO). 2 Over the past decade, insights into the molecular basis of gliomas have significantly improved both our biological understanding of glioma as well as our abilities to estimate their prognosis and their likelihood of response to specific therapies. 3 Recent studies have illustrated a unique genomic picture of grade II and III gliomas that showed distinct molecular lesions from those typically found in glioblastoma (GBM). 4,5 These tumors have an extremely high frequency of IDH1/2 mutation, which is accompanied either by 1p and 19q co-deletion or by TP53 mutations with or without ATRX alteration, together with other mutations such as those involving CIC and FUBP1. 5 IDH mutations are rare in primary GBMs but occur in 80% of clinically diagnosed grade II and III gliomas. 6 1p/19q co-deletion is more frequent in grade II and III gliomas with an oligodendroglioma component (40% 70%). ATRX alteration has been observed in 60% 80% of grade II and grade III astrocytomas, but only in 5% of primary GBMs. 7 Despite their better prognosis, many cases of grade II and III gliomas eventually progress to more aggressive tumors or GBMs over years. 2 As the classifications of grade II/III gliomas based on the genetic alterations have recently emerged, it is now a trend to include molecular data into the standard diagnostic algorithm of glioma, which might support treatment strategy selection, therapeutic trial design, and clinical prognosis evaluation. TERT promoter mutation (TERTp-mut) is another molecular hallmark of glioma, occurring in 70% 80% of primary GBMs and about 70% of oligodendrogliomas but less frequently in grade II and III astrocytomas. 8 12 Mutations in 2 hotspots (chr5, 1 295 228 C. T and chr5, 1 295 250 C. T) can enhance TERT promoter activity. 13 TERTp-mut is associated with a poor clinical outcome of glioblastomas; 8,14 however, its clinical value and biological characteristics remain elusive in grade II and III gliomas. In this study, we investigated the prevalence and prognostic impact of TERTp-mut in a series of 377 patients with grade II and III gliomas. TERTp-mut in grade II and III gliomas allows the opportunity to supplement histopathological classification with genomics, particularly when combined with the IDH mutational status. We evaluated the frequencies of molecular markers and biological processes characteristic in grade II andiiigliomaswithinthe4subgroupsbasedonidh and TERT promoter mutational status and defined a more convenient and accurate classification to aid clinicians in evaluating patient prognosis, designing therapeutic trials, and selecting the best treatment strategy. Materials and Methods Clinical and Molecular Characteristics of Samples Totally, 377 samples were used in our study (Supplementary material, Table S1). The whole transcriptome profile of 154 of these samples was obtained from the Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn) and included RNA sequencing data for 97 samples and mrna expression array data for 57 samples. 15,16 All patients had undergone surgical resection and postoperative adjuvant temozolomide radiotherapy. Patients were eligible for the study if their diagnosis was established histologically by 2 neuropathologists according to the 2007 WHO classification guidelines. 2 Tumor tissue samples were obtained via surgical resection. Samples with at least 80% tumor cells were selected for analysis. Survival data were collected by clinics during patient visits and/or phone interviews. All patients provided written informed consent, and the study was approved by the ethics committees of the participating institutes. The mutational status of IDH1/2 and TP53 and the methylation status of the MGMT promoter were determined using pyrosequencing or Sanger sequencing. 17,18 TERT Promoter Mutation TERT promoter mutational status was determined using Sanger sequencing. 14 Sequences covering mutational hotspots in the TERT core promoter (nucleotide numbers 1 295 228 [C228T] and 1 295 250 [C250T] from the human reference sequence (grch37 February 2009; http://genome.ucsc.edu/) were amplified using nested PCR. The primer sequences for the first PCR (primer set 1) were 5 -GTC CTG CCC CTT CAC CTT-3 (forward) and 5 -GCA CCT CGC GGT AGT GG-3 (reverse), which yielded a 273-bp product. PCR was performed in a total volume of 10 ml, consisting of 1 ml ofdnainsolution(10 50ng/mL), Platinum Taq DNA polymerase (1 unit), 1 ml of 10X PCR buffer (Invitrogen), 1.0 mm MgCl 2,0.1mMofeachdNTP,1%(v/v) DMSO, and primers (0.25 mm each). The first PCR was performed using a C1000 thermal cycler (BIORAD) with an initial denaturing step at 958C for 3 minutes, followed by 40 cycles of denaturation at 968C for 15 seconds, annealing at 628C for 20 seconds, extension at 728C for 30 seconds, and a final extension at 728C for 10 minutes. Primers for the nested PCR (Primer set 2) were 5 -CCG TCC TGC CCC TTC ACC-3 (forward) and 5 -GGG CCG CGG AAA GGA AG-3 (reverse) and yielded a 128-bp product. Nested PCR was performed using the same conditions as the first PCR, with the PCR product from the first reaction diluted 50 times as a template. Products from the second PCR were purified using illustra ExoProStar (GE Healthcare) and subjected to direct sequencing on an ABI 3100 PRISM DNA sequencer with the Big- Dye Terminator cycle sequencing kit (Applied Biosystems). The quality of all PCR products was checked via electrophoresis on 2% agarose gels before sequencing. 1p/19q Co-deletion Representative tumor areas were marked on hematoxylin and eosin-stained sections. The corresponding areas were identified on paraffin blocks, and new 4 mm sections were prepared. The material was deparaffinized with xylene, incubated with 0.3% pepsin in 10 mm HCl at 378C for 10 minutes, and denatured at 858C for 10 minutes. Dual-color fluorescence in situ hybridization was performed using LSI probe sets for 1p36/1q25 and 19q13/19p13 (spectrum orange-labeled 1p36 and 19q13 probes; spectrum green-labeled 1q25 and 19p13 probes; and Vysis), and evaluated in at least 200 nonoverlapping nuclei with intact morphology. 1100

Immunohistochemistry for Ki-67/p-MET/MMP-9 Immunohistochemistry was performed to detect Ki-67/p-MET/ MMP-9 protein expression according to the manufacturer s protocol. Anti-Ki-67/p-MET/MMP-9 antibodies (Santa Cruz Biotechnology) were used at a dilution of 1:100. Each slide was individually reviewed and scored by 2 independent neuropathologists. Staining was scored using a 4-point scale from 0 to 3, with 0 if there was no staining or very little staining, 1 if 10% of cells stained positively, 2 if 10% 30% of cells stained positively, and 3 if.30% of cells stained positively. Controls without primary antibody and positive control tissues were included in all experiments to ensure the quality of staining. 16,19,20 Statistical Analysis For the molecular subtype annotation, we applied prediction analysis of microarrays. 21 Gene ontology (GO) analysis of differentially expressed genes was performed using GeneCodis (http:// genecodis.cnb.csic.es/). 22,23 Kaplan-Meier survival analysis was used to estimate survival distributions. Overall survival (OS) was defined as the time interval between the date of surgery and the date of death. The log-rank test was used to assess statistical significance between stratified survival groups by GraphPad Prism version 4.0 statistical software. A gene was considered differentially expressed if the associated P value was,.05 and the mean absolute fold-change was 2. Chi-square and Fisher exact tests and Cox proportional hazard regression analyses were performed using SPSS version 13.0 software for Windows (SPSS). All differences were considered statistically significant at P,.05. Results Patient Characteristics and Treatment We identified patients with grade II and III grade gliomas, who underwent surgical resection at the Glioma Treatment Center Fig. 1. Survival of the 4 subgroups stratified according to IDH and TERT promoter mutations. TERTp-mut was a distinct prognostic factor in both the IDH-mut and IDH-wt subgroups. Survival analysis based on IDH and TERT promoter mutational status demonstrated a remarkable stratification (IDH-mut/TERTp-mut versus IDH-mut/TERTp-wt, P,.0001; IDH-mut/TERTp-wt versus IDH-wt/TERTp-wt, P ¼.0317; IDH-wt/ TERTp-mut vs. IDH-wt/TERTp-mut, P ¼.0495). of the Beijing Tiantan Hospital and the Beijing Sanbo Brain Hospital between January 2006 and July 2012. The study group comprised 377 patients (219 men, 158 women; age range, 18275 y; median age, 38 y) with WHO grade II or III disease. TERT promoter and IDH mutational status were detected for all 377 patients (Supplementary material, Table S1). The median preoperative KPS score was 90 (range, 40 100), and 314 patients (83%) scored.80. The IDH-mut subgroup showed markedly better preoperative KPS scores than the IDH-wt subgroup (P ¼.012). IDH mutations were common in astrocytomas (73.0%), oligoastrocytomas (75.3%) and oligodendrogliomas (84.3%). The IDH-mut subgroup more frequently had MGMTpmeth (P,.001) and TP53-muts (P ¼.004) than the IDH-wt subgroup. TERT promoter mutations prevalently occurred in oligoastrocytomas (51.1%) and oligodendrogliomas (72.5%) compared with astrocytomas (11.5%, P,.0001). These 62 gliomas with IDH mutation and 1p/19q codeletion were analyzed for an association with TERT promoter mutation (P,.0001). TERTp-mut i\is an Independent Predictor of Good Prognosis for Patients With Grade II and III Gliomas Among patients with grade II and III gliomas, those with IDHmut tumors showed significantly longer OS compared with patients with IDH-wt tumors (P,.0001, Supplementary material, Fig. S1A); patients with TERTp-mut tumors showed significantly longer OS compared with patients without TERTp-mut tumors (P ¼.0084, Supplementary material, Fig. S1B). In patients with WHO grade II gliomas, both IDH-mut and TERTp-mut were statistically significant factors for longer OS in Kaplan- Meier survival analysis (P ¼.0255, Supplementary material, Fig. S2A, and P ¼.0001, Supplementary material, Fig. S2B). However, for WHO grade III gliomas, IDH-mut was the only significant predictive factor for a good prognosis (P,.0001, Supplementary material, Fig. S2C); TERTp-mut had no significant prognostic value (P ¼.754, Supplementary material, Fig. S2D). We then tested the following factors as candidate variables in a multivariate Cox proportional hazards regression model analysis: age at diagnosis, KPS, IDH-mut, and TERTp-mut. TERTp-mut was an independent prognostic factor for OS in grade II and III gliomas after correcting for age, KPS, and IDHmut (hazard ratio [HR], 0.621, 95% confidence interval [CI], 0.388 0.995, P ¼.048, Supplementary material, Table S2). TERTp-mut i\is Associated with a Specific Prognosis According to the IDH Mutational Status IDH-mut was a significant prognostic factor for WHO grade II and III gliomas. We therefore sought to determine whether TERTp-mut could explain the better outcomes for patients with IDH-mut tumors compared with those having IDH-wt tumors. Interestingly, in stratifying this population according to IDH status, we found that the TERTp-mut was a distinct prognostic factor for both the IDH-mut and IDH-wt subgroups. TERTp-mut was associated with longer OS in the IDH-mut subgroup (P,.001, Supplementary material, Fig. S3A) but was closely associated with shorter OS in the IDH-wt subgroup (P ¼.0495, Supplementary material, Fig. S3B). Moreover, for WHO grade II tumors carrying IDH-mut, TERTp-mut was associated with longer OS (TERTp-mut 95 vs TERTp-wt 93 months, Neuro-Oncology 1101

P ¼.0015, Supplementary material, Fig. S4A), but for WHO grade II tumors with IDH-wt and III tumors with either IDH status, we found that TERTp-mut had no prognostic significance (Supplementary material, Figs. S4B D). Next,weinputtedage,KPS,andTERTp-mut as candidate variables into the multivariate Cox proportional hazards regression model analysis of the IDH-mut and IDH-wt subgroups, respectively. TERTp-mut appeared as an independent prognostic factor for OS in the IDH-mut subgroup after correcting for age and KPS (HR,0.444, 95% CI, 0.227 0.869, P ¼.018, Supplementary material, Table S3), but not in the IDH-wt subgroup (Supplementary material, Table S4). As shown in Fig. 1, survival analysis based on IDH and TERT promoter mutational status also demonstrated remarkable stratification of the clinical course in the 4 molecular subgroups (IDH-mut/TERTp-mut vs mut/tertp-wt, P,.0001; IDH-mut/ TERTp-wt vs IDH-wt/TERTp-wt, P ¼.0317; and IDH-wt/TERTpmut vs IDH-wt/TERTp-mut, P ¼.0495). In our cohort, those patients with a TERTp-mut alone had the poorest prognosis (medianos,27.7mo;5ysurvivalrate,29%),whiletumors with only an IDH-mut were associated with a more favorable prognosis (median OS, 85.7 mo; year survival rate, 73%). Patients with tumors harboring mutations in both the TERT promoter and IDH also had the best prognosis (median OS, 95 mo; 5 y survival rate, 92%). This contrasted with patients who had tumors that were both TERTp-wt and IDH-wt, because they made up a unique clinical group with a short OS (median OS, 92 mo; 5 y survival rate, 54%) (Supplementary material, Table S5). Gene Functional and Molecular Analysis We next evaluated the frequencies of known molecular markers in gliomas within the 4 subgroups. The distribution of genetic alterations as well as clinical factors in all 377 grade II and III gliomas are listed in Table 1. Interestingly, TP53-mut, a classic molecular feature of astrocytic tumors, occurred mutually exclusive in tumors with TERTp-mut (P,.001, Fisher exact probability test, 2-tailed). Furthermore, TERTp-mut was associated with the 1p/19q co-deletion (P ¼.002, Fisher exact probability test, 2-tailed) and oligodendroglial histology (128 of 229, 56%). Although there is no association between TERTp-mut and IDHmut in the whole group of grade II and III gliomas, the 2 genetic alternations always co-occur in oligodendroglial tumors (79.1%, P ¼.0278, Fisher exact probability test, 2-tailed). To identify differential biological processes in these subgroups, we obtained the whole transcriptome sequencing data of 97 samples from the CGGA and then screened for differentially expressed genes (P,.05, fold-change 2). In IDH-mut tumors, 123 and 84 genes were highly expressed in TERTp-mut and TERTp-wt groups, respectively, while in IDH-wt tumors, 124 and75geneswerehighlyexpressedinthetertp-mut and TERTp-wt groups, respectively. Supplementary material, Fig. A showed a heat map of the differentially expressed genes in these 4 subgroups. GO analysis showed that each molecular subgroup had a distinct pattern of biological processes from the GeneCodis database (Supplementary material, Fig. S5B). In IDH-wt tumors, TERTp-mut was associated with cell proliferation and DNA repair, while TERTp-wt was associated with a positive regulation of response to cytokine stimulus, endothelial cell-cell adhesion, positive regulation of lymphocyte proliferation, and glial cell migration. We also performed immunohistochemistry to analyze the expression of Ki-67, p-met, and MMP-9, which are markers of tumor proliferation and invasion (Fig. 2A). We observed that Ki-67 protein expression was significantly higher in IDH-wt/ TERTp-mut tumors compared with other groups (P,.001) and that p-met expression was the highest in IDH-wt/TERTp-wt tumors (P,.01). MMP-9 protein expression was the highest in IDH-wt/TERTp-mut tumors, although IDH-mut/TERTp-wt and IDH-wt/TERTp-wt tumors also had high expression levels of this protein (P,.05) (Fig. 2B). These results suggested that IDH-wt and TERTp-mut tumors have a greater capacity for cell proliferation, while IDH-wt and TERTp-wt tumors have more pronounced cell invasion and migration characteristics. Clinically Relevant and Biological Classification of Grade II and III Gliomas In order to help place our classification based on IDH and TERT promoter mutational status into the context of previous glioma classification systems, we used the gene expression signature described by The Cancer Genome Atlas (TCGA) to classify 154 of the above tumors with available transcriptome data into 1 of 4 gene expression subtypes: proneural, neural, classical, and mesenchymal (Fig. 3). As expected, proneural tumors enriched in the IDHmut subgroup (P,.001) showed MGMT methylation phenotype (Fig. 2B). These tumors with an oligodendroglioma component were more common in the IDH- TERTpmut cluster (P,.001) and were associated with the best survival; in contrast, however, IDH-mut/TERTp-wt tumors consisted predominantly of pure astrocytoma (P,.001) but were also associated with longer survival (Fig. 1). 1p/19 co-deletion and TP53 mutations were, respectively, the main genetic features in IDH-mut/TERTp-mut and IDH-mut/TERTp-wt clusters (P,.001 for both, Fig. 2B). Consistent with other groups reports, 24 classical gene expression was mostly restricted to the IDH-wt/TERTp-mut cluster (P,.001), which showed higher Ki-67 expression, evidence of a cell proliferation phenotype, and was predictive of poor survival. The IDH-wt/ TERTp-wt cluster was a heterogeneous mixture of the 4 TCGA subtypes, although the mesenchymal expression phenotype was predominant (P,.001), and the phosphorylation level of MET (a mesenchymal marker 25,26 ) was the highest in IDH-wt/ TERTp-wt tumors associated with the worse survival outcome. A simplified graphical summary of the key molecular and biological characteristics of the grade II and III glioma subgroups, as identified by our integrated classification strategy, is given in Fig. 3. Discussion In our study, we evaluated the clinical significance of TERTpmut in grade II/III gliomas. The strong association between TERTp-mut and oligodendroglial histology and the 1p/19q co-deletion suggests that this activating mutation is involved in oligodendroglial oncogenesis. 8,12,13,27 In the present study, 1p/q9q codeletion occurred in 34.7% of oligodendrogliomas (WHO grades II and III) and 31.3% of oligoastrocytomas (WHO grades II and III). This result was similar to that of our 1102

Table 1. Clinical and molecular characters of 377 lower grade glioma patients based on IDH and TERT promoter mutational status Variables Total (n ¼ 377) IDH/TERTpmut (n ¼ 117) IDHmut only (n ¼ 168) TERTpmut only (n ¼ 28) IDH/TERTpwt (n ¼ 64) P Value Median age (range, y) 38 (23 61) 37 (18 66) 45 (24 75) 38 (18 61),.0001 Age 38 y old 194 59 96 7 32.0175.38 y old 183 58 72 21 32 Sex Male 219 65 97 19 38.692 Female 158 52 71 9 26 Histology A 127 8 94 2 23,.0001 AA 21 1 5 6 9 O 39 26 6 3 4 AO 12 8 3 0 1 OA 133 67 44 5 17 AOA 45 7 16 12 10 Preoperative KPS score 80 314 100 146 20 48.0467,80 63 17 22 8 16 1p/19q codeleted status Yes 76 40 22 5 9.0003 No 249 66 123 18 42 NA 52 11 23 5 13 MGMT promoter methylation Methylation 73 31 29 5 8,.0001 Unmethylation 50 5 18 11 16 NA 254 81 121 12 40 TP53 mutation Mutation 82 2 68 4 8,.0001 Wild-type 189 87 43 19 40 NA 106 28 57 5 16 Resection Gross ttal 192 55 89 13 35.6617 Subtotal 185 62 79 15 29 Postoperativetherapy RT plus TMZ 142 31 57 21 33,.0001 RT alone 235 86 111 7 31 Abbreviations: A, astrocytoma; O, oligodendroglioma; OA, oligoastrocytoma; AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; AOA, anaplastic oligoastrocytoma; IDH, isocitrate dehydrogenase; TERT, telomerase reverse transcriptase; TP53, tumor protein p53; 1p/19q, chromosome arm 1p and 19q; MGMT, O6-methylguanine-DNA methyltransferase; KPS, Karnofsky performance score; RT, radiotherapy; TMZ, temozolomide. previous reports 28 32 but was unusual compared with other countries and other centers in China, 27,33,34 possibly because of the methodology and ethnic difference or the sample size. Our present large-scale study was based on the Han Chinese population and analyzed clinical and molecular pathological characteristics in patients with lower grade gliomas. However, TERTp-mut was more frequently detected in oligodendroglial tumors and occurred mutually exclusive in tumors with TP53-mut, a classic molecular feature of astrocytic tumors. We found that TERTp-mut was an independent predictive factor for a good outcome in patients with grade II and III gliomas, 70% 90% of whom carried IDH mutations 35 (consistent with previous findings). 27,36,37 Several other studies showed that TERTp-mut tended to occur more often in primary GBM with poor outcomes, 37,38 80% 95% of which remained IDH-wt. 6,17 Importantly, we also found that TERTp-mut gave opposite prognostic indications in IDH-mut and IDH-wt grade II/III tumors. Although TERTp-mut was not an independent factor after adjusting for age at diagnosis and preoperative KPS, it tended to occur more commonly in IDH-wt grade II/III tumors of patients who survived for a shorter time (HR, 1.437). This finding might reflect the previous observation that the TERTp-mut frequently occurs in primary glioblastoma lacking IDH-mut, which has been associated with poor prognosis. 8,12 In 2007, our team demonstrated that transfection with antisense-htert adenoviruses significantly inhibited human Neuro-Oncology 1103

Fig. 2. Key molecular aberrations of grade II/III glioma subgroups. (A) Representative photomicrographs of tumor sections following immunohistochemical analysis for Ki-67, p-met, and MMP-9 expression ( 200). (B) MGMT methylation was enriched in IDH-mut tumors; TP53 mutations mainly occurred in IDH-mut/TERTp-wt tumors, while 1p/19q codeletion dominated in IDH-mut/TERTp-mut; IDH-wt/TERTp-mut tumors and IDH-wt/TERTp-wt tumors expressed high Ki-67 and p-met, respectively. glioma cell proliferation in vitro and glioma growth in a xenograft mouse model, 39 concurrent with the finding that the TERTp-mut was associated with poor survival in patients with IDH-wt gliomas. We will further evaluate the prognostic value of TERTp-mut in a larger cohort of IDH-wt grade II/III gliomas. Given the prognostic differences between any 2 subgroups in this classification, we obtained whole transcriptome sequencing data from 97 samples to search for specific and differential biological processes between them. We did not find any distinct biological processes between IDH-mut/TERTp-mut 1104

Fig. 3. Graphical summary of key molecular and biological characteristics of grade II/III glioma subgroups. Based on the IDH and TERT promoter mutational status, grade II/III gliomas were stratified into 4 distinct prognosis subgroups. 1p/19q codeletion mainly occurred in the gliomas with both IDH and TERT promoter mutation, which had an oligodendroglial phenotype. Ki-67 protein expression was significantly high in IDH-wt/ TERTp-mut tumors ( 200). MET, a mesenchymal marker, was phosphorylated in IDH-wt/TERTp-wt tumors ( 200). Proneural gene expression phenotype enriched in the IDH-mut subgroup, and these tumors with an oligodendroglioma component were more common in the IDH-mut/ TERTp-mut cluster, while in contrast, IDH-mut/TERTp-wt tumors consisted predominantly of pure astrocytoma. Classical gene expression was mostly restricted to the IDH-wt/TERTp-mut cluster, which showed higher Ki-67 expression. The IDH-wt/TERTp-wt cluster was a heterogeneous mixture of the 4 TCGA subtypes, although the mesenchymal expression phenotype was predominant and the phosphorylation level of MET was the highest in IDH-wt/TERTp-wt tumors associated with the worse survival outcome. and IDH-mut/TERTp-wt tumors, both of which primarily showed increases in development and cell differentiation pathways. In IDH-wt tumors, TERTp-mut specific genes were more frequently thoseassociatedwithpathwaysincancer,cellproliferation, and DNA repair, while TERTp-wt was associated with the positive regulation of response to cytokine stimulus, endothelial cell-cell adhesion, positive regulation of lymphocyte proliferation, and glial cell migration. In our cohort, all patients were treated with postoperative radiotherapy, and some patients received adjuvant temozolomide therapy. Patients with IDH-wt/ TERTp-mut tumors more frequently had a poor outcome and increased DNA repair, which may explain the poor response to radiation and alkylating agent. These findings may suggest that these patients require more aggressive treatment, similar to that used for primary GBM patients. 40,41 We went on to detect 3 well-established markers, Ki-67, p-met, and MMP-9, which reflected the biological behavior of tumors. Ki-67 protein expression was significantly higher in IDH-wt/TERTp-mut tumors, indicating that this type of grade II and III glioma has a greater capacity for cell proliferation. p-met expression was the highest in IDH-wt/TERTp-wt tumors characterized by a greater degree of cell invasion and migration. Our study is the first to explore the biological characteristics and molecular aberrations of these 4 subgroups in grade II/ III gliomas based on IDH and TERT promoter mutational status. 27,37 We have also identified 4 TCGA subtype signatures based on the whole transcriptome sequencing data of 97 samples and mrna expression array data from 57 samples. We found that proneural expression signatures were enriched in the IDH mutation subgroup, among which the IDH-mut/TERTp-mut cluster had the highest frequency of the 1p/19q co-deletion (mainly in oligodendroglial tumors). In contrast, the IDH-mut/TERTp-wt cluster showed a pure astrocytoma phenotype with a high Neuro-Oncology 1105

frequency of TP53 mutations. Classical gene expression was mostly restricted to the IDH-wt/TERTp-mut cluster. Weller et al also demonstrated that a classical glioblastoma-like profile associated with IDH1/2 wild-type status and anaplastic astrocytic histology (corresponding to genomic group V of which most samples harbored TERT promoter mutations). 24 IDH-wt/ TERTp-wt cluster might be a heterogeneous entity as it included all 4 TCGA subtypes, although the mesenchymal expression phenotype was dominant. GO enrichment of specific genes from the IDH-wt/TERTp-wt cluster revealed increased activity of immune and inflammatory response pathways, similar to previous findings of preferential enrichment of both proinflammatory and immunosuppressive genes within the mesenchymal subtype. 42 We also observed that MET, an activator of the epithelial-mesenchymal transition, 43,44 was phosphorylated in IDH-wt/TERTp-wt tumors, further confirming the mesenchymal phenotype of this group, and providing a potential therapeutic target for MET inhibitors in these gliomas without obvious genetic aberrations. Several newly published studies 5,45,46 have validated prior reports that specific combinations of genetic alternations in IDH1/2, TERT, and co-deletion of 1p/19q, have the ability to reclassify gliomas into rational subsets and define gliomas biological and clinical behavior more accurately than stratifications based solely on histopathology. We also validated these 2 classification schemes (IDH-1p/19q-TERT, IDH-1p/19q) in our cohort (Supplementary material, Table S6 and S7); these 2 schemes could reflect the characteristics of molecular and clinical information in the lower-grade gliomas. Based on the IDH mutational and 1p/19q status algorithm, we observed that MGMT methylated tumors enriched in the IDH mutated tumors (57/70, 81.4%). Gliomas with TP53 mutations showed the IDH mutation, and none showed the 1p/19q codeletion phenotype (57/77, 74%). IDH mutated and 1p/19q codeleted gliomas (40/62, 64.5%) had a higher frequency of TERT promoter mutations compared with IDH mutated gliomas without 1p/19q codeletion (66/189, 34.9%) and gliomas with IDH wild type (28/92, 30.4%). In the IDH-1p/19q-TERT system for classification, 289 samples came into the 5 subgroups. Most oligodendroglial tumors were enriched in the triple-positive and IDH mutated, TERT mutated gliomas (66/161, 61.5%). In addition, triple-positive glioma had no pure astrocytic component tumors. Regardless of classification schemes, there was significant difference in overall survival of patients among these subgroups. In our cohort, the classification based on the IDH and TERT promoter mutational status might distinguish the different histology gliomas more clearly. On the other hand, these 4 groups had different ages at diagnosis, overall survival, and association with key biomarkers (p-met, MMP-9, and Ki-67), which might imply that they are characterized by distinct biological processes. In addition, we have compared the clinical characteristic and molecular alterations between lower-grade gliomas with TERT promoter mutations only and those without IDH and TERT promoter mutations (Supplementary material, Table S8). There was no significant difference in sex, preoperative KPS score, TP53 mutation, and MGMT promoter methylation between these 2 groups. However, there was a significant difference in age at diagnosis. Patients with TERT promoter mutations were considerably older than those without TERT promoter mutations in these IDH wild type samples, suggesting that TERT promoter mutations were associated with age of patients without IDH mutations. This is consistent with other teams reports 8,11 and complements the observed effect of inherited TERT variants on age at diagnosis in glioma patients. 47 Although TERT promoter mutations were not associated with morphology in IDH wild type gliomas, most gliomas without IDH and TERT promoter mutations only reached WHO II grade (P ¼.0053). Although our classification was different from the classifications from TCGA 45 and Mayo/UCSF/TCGA, 46 we hope that our scheme could provide a good knowledge of glioma molecular classification and better understanding for the classifications of TCGA 45 and Mayo/UCSF/TCGA. 46 In conclusion, the classification of grade II and III gliomas based on IDH and TERT promoter mutational status was more manageable and economical than other classifications based on gene expression profiles. It also reflected histological and morphology features, key molecular aberrations, and gene expression signatures, and could help to select more effective treatment strategy and monitor the treatment response. A further systematic analysis is needed to better delineate the role of the exact classification of IDH and TERT promoter mutations in the natural history of grade II and III gliomas and their response to treatment within a prospective randomized study design. Funding This work was supported by grants from National Key Technology Research and Development Program of the Ministry of Science and Technology of China (No. 2014BAI04B02), National High Technology Research and Development Program (No. 2012AA02A508), The Research Special Fund For Public Welfare Industry of Health (No. 201402008), Beijing Science and Technology Plan (No. Z131100006113018), International Science and Technology Cooperation Program (No. 2012DFA30470), National Natural Science Foundation of China (No. 91229121), and Special Fund Project of Translational Medicine in the Chinese-Russian Medical Research Center (No. CR201417). Conflict of interest statement. None declared. References 1. Dolecek TA, Propp JM, Stroup NE, Kruchko C. CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005 2009. Neuro Oncol. 2012;14(Suppl 5):v1 49. 2. Louis DN, Ohgaki H, Wiestler OD, et al. 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