{"paper_id":"b3e866de-61ba-48d5-8cc1-065aa8a19993","body_text":"Glioblastoma (GBM) is the most frequent and deadly primary brain tumor, accounting for approximately 45–50% of all primary malignant brain tumors [ 17 ,  18 ]. GBM is a heterogeneous entity, with a wide mutational spectrum. There has been an ever-increasing focus on molecular classification in GBM, to develop insights into the biology of this tumor and to subsequently improve diagnosis and treatment.\nTo emphasize the importance of molecular markers, the 2016 World Health Organization (WHO) revised neuropathological criteria identifies three categories of grade IV diffuse glioma. Two categories of GBM arise based on clustered genetic alterations, histologic variants, and clinical data [ 15 ],  IDH  wild-type and  IDH  mutant. An additional category of  H3F3A  K27 M mutant midline glioma has been designated grade IV, due the often poor prognosis of patients with these tumors. While  IDH  and  H3F3A  mutations identify gliomas with a distinct molecular origin, the remaining  IDH  wild-type subgroup of GBM, as it is defined currently, still contains significant heterogeneity. Emerging evidence indicates that  TERT  promoter ( TERT p) mutations, which are common in these tumors, could additionally be useful clinically to classify  IDH  wild-type GBMs into subgroups with specific clinical courses [ 7 ,  12 ].\nHere, we evaluated  TERT p wild-type ( TERT p-wt) GBMs to compare them to their  TERT p mutant counterpart GBMs. We performed sequencing on a broad panel of genes and evaluated for the presence of fusions in a cohort of GBMs, to evaluate the mutational profile of  TERT p-wt GBMs. In addition, we examined the clinical characteristics of this group.\n\nThe study was reviewed and approved by the human subjects’ institutional review boards of the Dana-Farber Cancer Institute and Massachusetts General Hospital (P10–454) and complied with HIPAA guidelines. We retrospectively reviewed the genomic database at our institution for adult GBM cases submitted for genotyping using the SNaPshot panel version 2. Demographic, treatment and follow-up data were retrospectively collected.\nSpecimens were subjected to genomic analysis utilizing SNaPshot 6 , a hybrid capture based method for single nucleotide variant (SNV) and insertion/deletion (indel) detection in tumor DNA. SNaPshot targets 108 genetic loci frequently mutated in 15 cancer genes, including  TERT  promoter,  IDH1 / 2 ,  TP53 ,  ATRX, PIK3CA, PIK3R1, NF1 , and  STAG2 . The detailed list of all genes included in the SNaPshot v2 panel is shown in Additional file  1 .\nExtracted tumor RNA were interrogated for fusions by the Archer® FusionPlex® Solid Tumor (AK0034) kit [ 25 ]. This technology utilizes an anchored multiplex polymerase chain reaction (AMP) technique that detects gene rearrangements in a fusion partner agnostic manner. FASTQ data analysis, including fusion calling, was performed by ArcherDx Analysis software v5.0.6 using default parameters. The detailed list of all genes included in Archer® FusionPlex® is shown in Additional file  1 .\nDNA was extracted from frozen tumor tissue and subjected to bisulfite treatment. Two separate methylation-specific PCR reactions were performed, one using primers specific for methylated  MGMT  promoter sequences, and a second using PCR primers specific for unmethylated  MGMT  promoter sequences [ 8 ].\nATRX immunohistochemistry was preformed using ATRX Cat # BSB-3295 from Bio SB. RTU (ready to use) pretreatment ER2 (EDTA ph 9.0) for 15 min. The clone BSB-108 was used, as previously reported [ 21 ].\nThe statistical association of  TERT p-wt GBM with other factors, including age, sex, other genomic alterations, and location of tumor, were analyzed using the Fisher exact test. The association of  TERT p-wt GBM with ATRX immunohistochemistry,  MGMT  promoter methylation status, and presence of fusion gene by solid fusion panel were each also evaluated. Cases with unavailable molecular or IHC data were excluded from the final correlation analysis.\nThe data were analyzed using the Fisher exact test. Description of overall survival (OS) was estimated by the Kaplan-Meier product limit method. A two-tailed  P  value of < 0.05 was considered to be statistically significant.\n\nWe identified 121 adult GBM cases with available molecular and immunohistochemistry data between 2016 and 2018 (Additional file  2 ). We excluded histologic GBMs containing  IDH  R132 and  H3F3A  mutations from statistical analyses ( n  = 11 and  n  = 1, respectively), for the reasons noted above [ 15 ].\nWithin this cohort ( n  = 109), the average age of patients was 60 years (range 18–84 years). Genetic alterations in the  TERT  gene were detected in 93 tumors; 92 were  TERT p mutant (84.4%), and an additional case had a  TERT-SUB  fusion. The remaining 16 patients (14.7%) had  TERT p-wt GBM (Fig.  1  and Additional file  2 ). The average age of patients with  TERT p-wt GBMs was 53.2 years, which was significantly younger than the average age of their counterparts with  TERT p mutant GBMs (60.7 years,  p  = 0.0096), and significantly older than the average age of patients with  IDH  mutant GBMs (38.6 years,  p  = 0.0041). Fig. 1 Flowchart depicting mutation breakdown of adult glioblastoma cases\nFlowchart depicting mutation breakdown of adult glioblastoma cases\nAcross the cohort of  IDH -wt GBM, the male to female ratio was 1.66.  TERT p-wt GBMs did manifest a numerically higher proportion of male patients (13/16, 87.5%), compared with 55/93 male patients (59%) with  TERT p mutant GBMs, but this difference was not statically significant ( p  = 0.103).\nWe examined the location of the primary tumor presentation. In the  TERT p-wt group, the primary tumors were mainly found in a supratentorial (13) and thalamic/midline location (1), but also in a cerebellar site (3 cases). In contrast, in the  TERT p mutant group, the tumors were exclusively located supratentorially (91) or thalamic/midline (2), with none found in the cerebellum. Consequently, a significant correlation between  TERT p-wt status and cerebellar location ( p  = 0.0027) was observed. Of note, one of the cerebellar GBMs occurred in a patient with a  NF1  germline mutation (Neurofibromatosis type 1).\nThe median time of follow-up in surviving patients was 189 days for the  TERT p-wt group and 246 days for the  TERT p mutant group. Due to the short follow-up time, survival analyses may be underpowered to detect differences. Nonetheless, no detectable difference in survival was observed between the two groups ( p  = 0.74).\nWe examined genetic and epigenetic correlations between  TERT p-wt versus mutant tumors. Four  TERT p mutant cases were found to harbor a hotspot  BRAF  V600E mutation, which is characteristic of epithelioid GBM [ 14 ].  NF1  mutations were more commonly seen in  TERT p-wt GBMs (6/16, 37.5%), in comparison with 18/93 (19%) in the  TERT p mutant GBM cohort, however, this was not a statistically significant difference ( p  = 0.11). Also, we did not observe a significant difference in  MGMT  promoter methylation status in the  TERT p-wt group vs. the mutant group (7/14 vs. 36/90,  p  = 0.56).\nActivating alterations in the PI3K pathway (mainly  PIK3CA  or  PIK3R1 ) were detected in 25 out of 109 cases in the cohort (23%) (Additional file  3 ). Interestingly, we observed a strong correlation between  TERT p-wt status and mutations targeting the PI3K pathway: 9/16 (56%) of  TERT p-wt GBMs contained a PI3K pathway alteration, while only 16/93 (17%) of mutant GBMs harbored these alterations ( p  = 0.0018) (Fig.  1 ). Furthermore, we detected an inverse correlation between  PIK3CA / PIK3R1  and  EGFR  alterations. Only 2/25 cases (8%) with a PI3K pathway alteration had an  EGFR  mutation or  EGFRvIII , whereas 38/82 of PI3K wild-type GBM had an  EGFR  alteration (46.3%,  p  = 0.0003).\nMoreover, as expected,  ATRX  mutations were detected by sequencing in 6/16 (37.5%)  TERT p-wt GBMs, while only 6/93 (6.5%) of  TERT p mutant GBMs had an  ATRX  mutation. Consequently, this manifested as a significant correlation between  TERT p-wt status and  ATRX  mutation ( p  = 0.0022). Of note, our workflow for assigning mutation was highly sensitive, leading to potential false positive assignments of  ATRX  candidate alterations that may not functionally inactivate the protein product. The further assessment of ATRX loss-of-expression using immunohistochemistry revealed a similarly significant result: 4/13 (31%) of  TERT p-wt GBMs had ATRX loss vs. 0/80 mutant GBMs ( p  = 0.0002) (Fig.  2 ). Fig. 2 Summary of clinical features and molecular alterations in  TERT p-wt glioblastoma\nSummary of clinical features and molecular alterations in  TERT p-wt glioblastoma\nFinally, we noted that 8/16 (50%) of  TERT p-wt GBMs harbored mutations in the BAF complex gene family ( SMARCA4 ,  SMARCB1 ,  ATRX , and  ARID1A ), compared with only 8/93 of  TERT p mutant GBMs ( p  = 0.0002). Given the role of  ATRX  in telomere maintenance, mutations in either group (ATRX vs SWI/SNF) may be unrelated. Nevertheless, we found that this association remained significant when excluding  ATRX  (3/16 (18.8%) of  TERT p-wt GBMs harboring mutations compared with only 2/93 of  TERT p mutant GBMs,  p  = 0.022). When combined with our analyses above, we detected a significant difference in co-occurrence between mutations in the BAF complex and PI3K pathway genes by comparing the  TERT p-wt ( n  = 5/16) and  TERT p mutant groups ( n  = 1/93,  p  = 0.0002) (Fig.  3 ). Fig. 3 Venn diagram depicting BAF complex mutation ( SMARCA4, SMARCB1, ATRX, or ARID1A ) and PI3K mutations relationship in  TERT  p-wt ( a ) and  TERT  p mutant ( b ) glioblastomas (GBM). Cases negative for both BAF complex and PI3K mutations amounted to  n  = 5 and  n  = 70, respectively\nVenn diagram depicting BAF complex mutation ( SMARCA4, SMARCB1, ATRX, or ARID1A ) and PI3K mutations relationship in  TERT  p-wt ( a ) and  TERT  p mutant ( b ) glioblastomas (GBM). Cases negative for both BAF complex and PI3K mutations amounted to  n  = 5 and  n  = 70, respectively\n\nThe WHO 2016 established an  IDH  wild-type subgroup of GBM, comprising the majority of adult grade IV gliomas, yet, this diagnostic grouping still contains significant heterogeneity. In an effort to better sub-classify  IDH -wt GBMs, we used a broad panel of genes to genotype a large cohort of these neoplasms. In our analyses, we show that the  TERT p-wt subgroup of  IDH -wt GBM contains a distinct clinical and molecular profile.\nOur findings should be interpreted in the context of extensive recent work studying adult high-grade gliomas. Over the last several years, a strong relationship has been demonstrated between mutational status and clinical, radiological, and molecular characteristics in adult diffuse gliomas [ 1 ,  2 ,  7 ]. Recently, Eckel-Passow et al. identified five main glioma molecular groups based on three alterations: 1p/19q co-deletion, and  TERT p and  IDH  mutations. The groups had different ages of onset, survival, and associations with germline variants [ 7 ]. In addition, Aibaidula et al. specifically examined the adult  IDH  wild-type lower-grade gliomas, demonstrating significant heterogeneity within this group, with differences in prognosis based on further molecular classification by biomarkers such as  TERT p mutation,  EGFR  amplification,  H3F3A  mutation, and  MYB  amplification [ 1 ]. Focusing on GBM, Arita et al. highlighted the importance of  TERT p mutation,  IDH  mutation, and  MGMT  promoter methylation status on prognosis [ 2 ]. Furthermore, Stichel et al. demonstrated the potential of  EGFR  amplification, combined chromosome 7 gain and chromosome 10 loss, and  TERT p mutations for classifying  IDH  wild-type GBM [ 24 ].\nTERT p mutant GBMs show increased telomerase activation due to the increased TERT expression. In comparison, it is well-established that  IDH  mutant astrocytic gliomas often display the characteristic phenotype termed “alternative lengthening of telomeres” or ALT, associated with mutations in  ATRX  [ 10 ,  11 ,  13 ]. Another study that attempted to further sub-classify the 5 integrated WHO glioma groups by  ATRX  and  TERT  promoter status showed that  ATRX  alterations were enriched in  TERT p-wt GBM [ 19 ]. A further study of the  TERT p-wt subgroup by Diplas et al. identified  SMARCAL1  as an additional mechanism of telomere maintenance within this subgroup [ 6 ].\nIn agreement with prior studies, we observed that  TERT p-wt patients are significantly younger than their  TERT p mutant counterparts [ 7 ] (Additional file  4 ). In addition, we identified a significantly higher rate of cerebellar GBM in the  TERT p-wt compared with the  TERT p mutant patients. Our finding is consistent with prior studies that have shown that cerebellar GBMs occur in patients that are younger than patients with supratentorial GBMs, and have decreased frequency in  TERT p mutations and more frequent  NF1  mutations [ 16 ,  20 ]. Taken together with our findings, these data support the proposal that cerebellar GBMs may comprise a distinct subclass of tumor, which may arise via an alternative molecular etiology when compared to supratentorial  TERT p mutant GBM.\nPI3K pathway alterations are frequently detected in gliomas, most commonly in grade IV lesions [ 4 ,  9 ]. Our data demonstrate that  TERT p-wt GBMs are significantly enriched for PI3K pathway mutations compared with  TERT p mutant GBM. Moreover, mutations in  ARID1A  and other components of the SWI/SNF chromatin remodeling complex (collectively known as the BAF complex), have been previously reported to be frequent in various cancer types (e.g. endometriosis-associated ovarian cancers, endometrial cancers and non-gynecological tumors) [ 3 ,  5 ,  22 ,  23 ]. Interestingly, in these cancers, alterations of gene encoding for components of the BAF complex frequently co-occur with activating mutations in  PIK3CA  [ 3 ,  23 ]. It has been additionally reported that dysregulation of the PI3K signaling pathway and loss of function of  ARID1A  may have a combination effect on tumor development [ 5 ,  22 ]. We speculate that this association may extend to a specific subset of gliomas, namely  TERT p-wt GBM cases, which we find are enriched for BAF complex alterations and activating mutations in genes within the PI3K pathway. Following the logic of WHO 2016 classification, our findings suggest the potential definition of a molecular subtype of high-grade glioma, with implications for the utilization of targeted therapy in these patients [ 22 ].\n\nIn conclusion, this study identifies frequent PI3K pathway and BAF complex genetic alterations as co-occurring hallmarks of  TERT p-wt GBM, potentially reflecting a unique molecular etiology of these tumors. If further validated, these findings may have significant implications for the sub-classification of  IDH -wt GBM. Optimal management of these patients remains to be defined, but at a minimum, our data suggest that  TERT p-mutant and  TERT p-wt GBMs should be analyzed separately in future clinical studies, as they likely comprise distinct subclasses of neoplastic disease.\n\nAdditional file 1: A detailed list of all genes included in the SNaPshot v2 panel. (DOCX 14 kb) \n Additional file 2: A table including patients’ and tumor characteristics. (XLSX 20 kb) \n Additional file 3: A table listing all detected PI3K alterations in the cohort. The majority of alterations were reported in COSMIC ( https://cancer.sanger.ac.uk/cosmic/browse/genome ) and/or occured at hospot locations in TumorPortal (http://www.tumorportal.org/). Reference human transcripts used: ENST00000263967.3 (PIK3CA) and ENST00000521381.1 (PIK3R1). (XLSX 11 kb) \n Additional file 4: Age distribution according to TERTp mutations. (TIF 462 kb)\nA detailed list of all genes included in the SNaPshot v2 panel. (DOCX 14 kb)\nA table including patients’ and tumor characteristics. (XLSX 20 kb)\nA table listing all detected PI3K alterations in the cohort. The majority of alterations were reported in COSMIC ( https://cancer.sanger.ac.uk/cosmic/browse/genome ) and/or occured at hospot locations in TumorPortal (http://www.tumorportal.org/). Reference human transcripts used: ENST00000263967.3 (PIK3CA) and ENST00000521381.1 (PIK3R1). (XLSX 11 kb)\nAge distribution according to TERTp mutations. (TIF 462 kb)","source_license":"CC-BY-4.0","license_restricted":false}