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Molecular landscape of IDH ‐wild type, pTERT ‐wild type adult glioblastomas

Abstract Telomerase reverse transcriptase (TERT) promoter (pTERT) mutation has often been described as a late event in gliomagenesis and it has been suggested as a prognostic biomarker in gliomas other than 1p19q codeleted tumors. However, the characteristics of isocitrate dehydrogenase (IDH) wild type (wt) (IDHwt), pTERTwt glioblastomas are not well known. We recruited 72 adult IDHwt, pTERTwt glioblastomas and performed methylation profiling, targeted sequencing, and fluorescence in situ hybridization (FISH) for TERT structural rearrangement and ALT (alternative lengthening of telomeres). There was no significant difference in overall survival (OS) between our cohort and a the Cancer Genome Atlas (TCGA) cohort of IDHwt, pTERT mutant (mut) glioblastomas, suggesting that pTERT mutation on its own is not a prognostic factor among IDHwt glioblastomas. Epigenetically, the tumors clustered into classic‐like (11%), mesenchymal‐like (32%), and LGm6‐glioblastoma (GBM) (57%), the latter far exceeding the corresponding proportion seen in the TCGA cohort of IDHwt, pTERTmut glioblastomas. LGm6‐GBM‐clustered tumors were enriched for platelet derived growth factor receptor alpha (PDGFRA) amplification or mutation (p = 0.008), and contained far fewer epidermal growth factor receptor (EGFR) amplification (p < 0.01), 10p loss (p = 0.001) and 10q loss (p < 0.001) compared with cases not clustered to this group. LGm6‐GBM cases predominantly showed ALT (p = 0.038). In the whole cohort, only 35% cases showed EGFR amplification and no case showed combined chromosome +7/−10. Since the cases were already pTERTwt, so the three molecular properties of EGFR amplification, +7/−10, and pTERT mutation may not cover all IDHwt glioblastomas. Instead, EGFR and PDGFRA amplifications covered 67% and together with their mutations covered 71% of cases of this cohort. Homozygous deletion of cyclin dependent kinase inhibitor 2A ( CDKN2A)/B was associated with a worse OS (p = 0.031) and was an independent prognosticator in multivariate analysis (p = 0.032). In conclusion, adult IDHwt, pTERTwt glioblastomas show epigenetic clustering different from IDHwt, pTERTmut glioblastomas, and IDHwt glioblastomas which are pTERTwt may however not show EGFR amplification or +7/−10 in a significant proportion of cases. CDKN2A/B deletion is a poor prognostic biomarker in this group.

glioblastomas. Instead, EGFR and PDGFRA amplifications covered 67% and together with their mutations covered 71% of cases of this cohort. Homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A)/B was associated with a worse OS (p = 0.031) and was an independent prognosticator in multivariate analysis (p = 0.032). In conclusion, adult IDHwt, pTERTwt glioblastomas show epigenetic clustering different from IDHwt, pTERTmut glioblastomas, and IDHwt glioblastomas which are pTERTwt may however not show EGFR amplification or +7/À10 in a significant proportion of cases. CDKN2A/B deletion is a poor prognostic biomarker in this group.

| INTRODUCTION
Tumor cells may display telomere dysfunction and shortened telomeres that lead to chromosome instability. TERT (telomerase reverse transcriptase) activation regenerates telomeres sufficiently to maintain them above the critical threshold and stabilize the tumor genome [1]. TERT promoter (pTERT) mutation leads to telomerase activation, and increased TERT expression [2]. But pTERT mutation only accounts for about 30% of instances of TERT activation across different cancers, the other methods being structural variants and chromosomal rearrangements to enhancer elements or oncogene activation or other mechanisms [1]. And pTERT mutations are often found in cells of low rates of renewal like brain cells and are rare in malignancies with very high rates of proliferation [1].
About 70%-80% of IDHwt (wild type for isocitrate dehydrogenase) glioblastomas are pTERT mutant (mut) [2][3][4][5][6]. pTERT mutation is often regarded as the last stage of gliomagenesis when the tumor presents clinically by survival-promoting mutations [4,7]. pTERT mutation has been found to be inversely correlated with IDH in glioblastoma and therefore the incidence of pTERT promoter mutation is high in IDHwt glioblastoma and low in Grade 4 astrocytoma, IDH-mutant [2,[8][9][10]. This has also been our and others' experience where in, Grade 4 astrocytoma, IDH-mutant incidence of TERT promoter mutation is very low [11][12][13]. In Grade 4 astrocytoma, IDH-mutant, telomere maintenance is usually achieved by ALT (alternative lengthening of telomeres) that is typically associated with mutations of the gene ATRX (alpha thalassemia/mental retardation syndrome X-linked) [11,14,15]. Ceccarelli et al. showed that ATRX mutation, rather than pTERT mutation, is associated telomere length [16]. pTERT mutations are also described to be associated with a worse survival in some studies [17,18] but not with others [5,19] and ALT was associated with a better survival in glioblastomas [20].
For glioblastomas which are pTERTwt, there is relatively little information and the mechanism of telomere maintenance remains unknown. Here, the term "pTERTwt" is designated just for glioblastomas that are wildtype for the promoter of the gene TERT. To our knowledge, there were only two publications dedicated to IDHwt, pTERTwt glioblastomas. Diplas et al. identified SMARCAL1 inactivating mutation, in addition to ATRX mutation, as a genetic mechanism of ALT [3]. SMARCAL1 is also a member of the SW1/SNF family of chromatin remodelers like ATRX [26] and mutations of SMARCAL1 lead to ALT telomere maintenance [3]. The authors also identified another mechanism of telomerase activation in glioblastomas via chromosomal rearrangements of TERT. The location of breakpoint was variable but the translocations led to increased TERT expression. Next-generation sequencing was performed in this study in some of the IDHwt, pTERTwt glioblastomas in this publication but methylation profiling was not performed [3]. Williams et al. studied a small series 16 cases and found them to be from younger patients than pTERTmut glioblastoma patients and they showed frequent P13K pathway mutations [6].
In this study, we examined a cohort of IDHwt, pTERTwt adult glioblastomas with methylation profiling, DNA-targeted sequencing, fluorescence in situ hybridization (FISH) for ALT (alternative lengthening of telomeres), and FISH for TERT structural rearrangement. We aim to provide a better understanding of the molecular landscape of IDHwt, pTERTwt glioblastomas and to identify genomic or genetic tests which can help risk stratification.

| Patients
We recruited unselectively adult IDHwt glioblastomas (aged 18 or above) from the archives of the neurosurgical teams at the Prince of Wales Hospital, Chinese University of Hong Kong from April 2013 to March 2020, and Hua Shan Hospital, Fudan University, Shanghai, from January 2015 to December 2017. The reasons for the difference in years between the two institutions was the difference in annual caseloads for the two neurosurgical teams and we aimed to collect a cohort that was within the limits of our research resource capacity. Histology of all recruited cases was reviewed (Hong Chen and Ho-Keung Ng) to confirm histological features of glioblastoma as per WHO 2021 Classification, namely mitoses, cellular atypia, necrosis, and/or microvascular proliferation [27]. The approach to gliomas in both hospitals was maximal safe resection in the first instance. Ethics approvals were obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee, and the Ethics Committees of Huashan Hospital, Fudan University, Shanghai. Data on patient demographics and treatments were retrieved from institutional paper and electronic records. Survival data were obtained from follow-up clinic visits and direct contact with patients or close relatives via phone.

| Sanger sequencing of IDH1 and IDH2
The IDH status of the 72 glioblastomas in this study was first identified in our routine diagnostic practice. At our hospitals, IDH status of determined by Sanger sequencing for the codon 132 of IDH1 and codons 140 and 172 of IDH2. As described below, these cases were further evaluated with target sequencing in which the panel was designed to examine all coding sequences of IDH1 and IDH2 genes.

| TERT promoter mutation analysis
TERT promoter mutations were evaluated by Sanger sequencing according to our previous publication [28]. In brief, crude cell lysate was obtained from formalin-fixed paraffin-embedded (FFPE) sections. Nucleic acid in the cell lysate was amplified using forward primer, reverse primer, and KAPA HiFi HotStart ReadyMix (Sigma). The forward primer was 5 0 -GTCCTGCCCCTTCA CCTT-3 0 and the reverse primer was 5 0 -CAGCGCTGC CTGAAACTC-3 0 . PCR products were then purified and sequenced with BigDye Terminator Cycle Sequencing kit (Life Technologies).

| Illumina infinium methylation EPIC BeadChip array
FFPE sections were sent to Sinotech Genomics Co., Ltd, Shanghai, where the DNA was extracted, bisulfite modified, and subjected to DNA methylation profiling by EPIC Illumina Infinium Human (850 k) array.

| DNA methylation pre-processing
We performed noob (normal-exponential convolution using out-of-band probes) background correction [29] and dye bias correction using the minfi package [30]. The DNA methylation score for each locus was presented as a beta (β) value (β = (M/[M + U])), where M and U indicated the mean methylated and unmethylated signal intensities for each locus, respectively. β-values ranged from zero to one, with a score of zero indicating no DNA methylation and a score of one indicating complete DNA methylation. A detection p-value also accompanied each data point and compared the signal intensity difference between the analytical probes and a set of negative control probes on the array. Any data point with a corresponding p-value >0.05 was deemed not to be statistically significantly different from the background and was thus marked as "NA".

| Classification of glioma samples based on TCGA DNA methylation subtypes
Samples were classified into the previously published glioma IDHwt molecular subtypes (classic-like, mesenchymal-like, and LGm6-glioblastoma [GBM]) using the CpG methylation signatures and method previously described [16]. We used a predictive model to classify the samples into one of the three IDHwt molecular subtypes, using the R packages and the randomForest. Cases were assigned to the methylation groups according to the highest score.

| Identification of copy number variations with EPIC 850 k array
Assessment of copy number variations (CNVs) was performed according to our previous publication [11]. The "conumee" R package in Bioconductor (http://www. bioconductor.org/packages/release/bioc/html/conumee. html) was employed to evaluate CNVs. A log2 ratio AE 0.35 was used as the cutoff of amplification/loss and a log2 ratio À 0.415 was used as the cutoff for homozygous loss [31]. GISTIC v2.0 analysis was conducted to identify significantly recurrent copy number amplification/gain and deletions at arm-level and focal-level, defined as affected regions spanning less than 50% of a chromosome arm.

| Targeted sequencing
DNA was obtained from FFPE sections using GeneRead DNA FFPE kit (Qiagen). The quality and quantity of extracted DNA were evaluated by QIAseq DNA Quanti-MIZE Assay (Qiagen). Samples that passed quality control were subjected to library preparation with a custom QIAseq Targeted DNA Panel, which examined coding exons or hotspots of 91 glioma-relevant genes (Table S1). The DNA libraries were then further qualified before the libraries were sequenced with MiSeq v3 (Illumina).
Paired-end reads were aligned to the hg19 (GRCh37) build of the human reference genome with BWA-MEM algorithm on GeneGlobe platform (Qiagen). Variants were called using smCounter2 and annotated using wANNO-VAR. Variants with the following criteria were excluded: not passing quality filters, with variant allele fractions of ≤10%, with variant allele counts of ≤5, or with minor allele frequencies of >1% in the overall human population or East Asians or documented in public databases (1000 Genomes, ExAc, gnomAD exome, and genome databases). Mutation events were checked with the Catalog of Somatic Mutations in Cancer (COSMIC) as per other previous studies on brain tumors [32][33][34][35].

| TERT structural rearrangement by FISH
TERT rearrangement was evaluated by FISH. FISH break-apart probes were designed according to previous publication [3]. In brief, 4-μm thick FFPE sections were deparaffinized in xylene, treated with 1 M sodium thiocyanate, digested in pepsin solution, rinsed in milli-Q water and dehydrated. The labeled probes were denatured and hybridized to the section overnight. Sections were then washed, stained with Vectashield mounting medium, and visualized under a Zeiss Axioplan fluorescence microscope. Samples were considered positive when break-apart signal was noticed in >5% of evaluated nuclei [3].

| Telomere-specific FISH analysis
ALT phenotype was examined with the Telomere PNA FISH kit (K532511, Dako) on 4-μm thickness FFPE sections. Tumor areas on the slides were identified and marked for evaluation. ALT-positive cases were identified when ≥5% of tumor cells exhibited large, very bright intranuclear foci of telomere FISH signals [36,37]. Endothelial cell nuclei were used for normal internal control purposes.

| TCGA clinical data
Clinical data of a total of 326 glioblastoma IDHwt, pTERTmut samples with available survival data were downloaded from The Cancer Genome Atlas (TCGA) Data Portal using TCGAbiolinks package (v2.18) function and imported into R (https://www.r-project.org) for further analysis [38]. All cases were aged above 18.

| Statistical analysis
Statistical analysis was performed on IBM SPSS software. Overall survival (OS) was defined as the period of time between operation and death or the last follow-up. Chi-square or Fisher's test was applied to determine correlation between molecular alterations and clinical parameters and between different molecular alterations. Survival curves were evaluated by the Kaplan-Meier (KM) method, and log-rank test was done to compare survival distribution between groups. Multivariate analysis was performed by Cox proportional hazards model. A p < 0.05 (two-sided) value was considered statistically significant.

| Characteristics of the study cohort
During the period described, 285 cases of IDHwt adult glioblastomas were retrieved from the two neurosurgical teams for review and sequencing. By Sanger sequencing, 72 cases of IDHwt, pTERTwt glioblastomas were identified and retrieved for this study. Methylation profiling was performed in all 72 cases. FISH for ALT and TERT structural rearrangement were performed in 71 cases due to lack of materials. DNA targeted sequencing was unsuccessful in 5 cases, due to small quantity of DNA available.
A summary of the molecular findings in this cohort, including CNVs and different means of telomere maintenance is shown in Figure 1. Intensity data (IDAT) of the 72 cases is uploaded at https://www.surgery.cuhk.edu.hk/ btc/hsbc/.

|
LGm6-GBM subtype of the TCGA classification is the main group within IDHwt, pTERTwt glioblastomas, and TERT promoter mutation was not a prognosticator when compared with TCGA The clinical profile of the cohort is presented in Table 1. The mean and median of this cohort were 51.9 and 57 years old, respectively. Male to female ratio was 1.25:1. Most tumors were located in the hemisphere.
About 65% of the patients underwent gross total resection as evaluated by post-operation MR imaging. Chemotherapy and radiotherapy were given to 78% and 72% of the patients. OS and progression-free survival (PFS) data were available in 98.6% and 87.5%, respectively. The mean and median OS of the cohort were 18.4 and 15 months, respectively. Univariate analysis revealed that gross total resection, chemotherapy, and radiotherapy were the prognosticators for OS and PFS ( Table 2).
We compared the OS of this cohort with the cohort of the adult IDHwt, pTERTmut glioblastomas (n = 326) retrieved from TCGA and found no significant difference in OS between the two cohorts ( Figure 2A). This suggests that pTERT mutation on its own is not a prognostic factor in IDHwt glioblastomas. TCGA only contained five cases of pTERTwt glioblastomas and they were all IDHmut. Interestingly, the mean age of the TCGA IDHwt, pTERTmut glioblastomas is 61.8 years old and is significantly higher than the mean age of this present IDHwt, pTERTwt cohort of 51.94 years old (p = 0.001).
LGm6-GBM cases and non-LGm6-GBM cases in this cohort were also different in their prevalence with MDM4 and RB1 alterations (Table 2).
3.3 | The mechanism of telomere maintenance of a proportion of IDHwt, pTERTwt glioblastomas remains unaccounted for For the mechanism of telomere maintenance, we performed FISH to detect TERT structural rearrangement and ALT in all samples with the exception of one case lacking sufficient tissue (Figures 3A-D).
Overall, 44 cases (62%) showed ALT, 4 cases showed ATRX mutations, 2 cases (3%) showed SMARCAL1 mutation, and 17 cases (24%) showed TERT structural rearrangement, and these cases overlapped ( Figure S2). The LGm6-GBM subtype was enriched with cases showing ALT (p = 0.038), and TERT rearrangement and ATRX/SMARCAL1 mutations were not associated with methylation subtypes. Nevertheless, with the exceptional of one case, all ATRX/SMARCAL1 mutations were detected in LGm6-GBM subtype. Similarly, all ATRX/ SMARCAL1 mutant, except one case, were positive for ALT. ATRX mutation and SMARCAL1 mutation were mutually exclusive. And TERT structural rearrangement was correlated with ATRX/SMARCAL1 mutations (p = 0.011). One-quarter of TERT-rearranged tumors carried ATRX/SMARCAL1 mutation, whereas only 4% of non-TERT-rearranged tumors carried ATRX/SMAR-CAL1 mutation. Most of the TERT-rearranged positive tumors were also positive for ALT (12/17, 71%). TERT rearrangement was not a prognosticator (Table S4). We did not make separate calculations for ATRX, SMAR-CAL1 mutations or TERT amplification as the numbers of cases for these groups were too small. PDGFRA amplification was enriched in cases showing ALT (20/44, 45%) compared with the non-ALT group (7/27, 26%). EGFR amplification was more frequent in ALT-negative cases (52%) than positive cases (25%, p = 0.021). For the whole cohort, 23 cases (32%) the mechanism of telomere maintenance was not accounted for by either ALT, TERT structural rearrangement or ATRX or SMARCAL1 mutations. This group of "mechanism of telomere maintenance not yet found" showed no association with any methylation subtypes and could be found in all of them. Interestingly, they showed an enrichment of EGFR amplification (52%, p = 0.033, Table S5).
DNA-targeted sequencing for 91 genes (Table S1) was successful in 67 (93.1%) cases. All mutations are listed in Table S7 and the more frequent mutations are shown in Figure 1. Diplas et al. found that 20% (5 cases) of their IDHwt, pTERTwt glioblastomas contained BRAFV600E mutation, a biomarker of potential therapeutic significance but we were only able to identify 2 such cases (3%) in this cohort. We did not detect a prognostic implication of single-gene mutations in the whole cohort and also among different methylation subtypes (Table S8). ATRX mutation was detected in 4/67 (6%) of the cases. All ATRX mutations were found in tumors of the LGm6-GBM subtype. ATRX mutation and chromosome +7 or À 10 were also found to be mutually exclusive. LGm6-GBM (frequency and number of cases) p-Value (non-LGm6-GBM vs. LGm6-GBM tumors) Overall, we detected 4.15 AE 6.187 mutations per sample. With the exception of one tumor, MMR (mismatch repair genes-MSH2, MSH6, MLH1, and PMS2) mutations were found in the LGm6-GBM tumors (17.5% vs. 3.7%). We performed sanger sequencing to confirm MMR mutations in 6 cases with enough materials to confirm the findings of NGS (Figures S4A-C). Review of the clinical history of these cases showed no other cancer and these tumors were all treatment naïve. The mean age of patients in this cohort (43.0 years old as mentioned above) was high for the possibility of a germline disease. As expected, cases positive for MMR mutations showed a higher mutation load than those that were negative (p = 0.017; 9.00 AE 11.832 mutations/sample vs 3.49 AE 4.794 mutations/sample). ATRX mutated cases showed a higher mutation load than those without (p < 0.001; 22.25 AE 13.022 mutations/sample vs. 3.00 AE 3.172 mutations/sample). A possible explanation is the presence of a link between ATRX deficiency and impaired non-homologous chromosomal end joining, rendering cells sensitive to DNA-damaging agents [39]. TERTrearrangement positive patients also carried higher number of mutations compared to the negative patients (p = 0.009; 7.63 AE 10.658 vs. 3.02 AE 3.396).

| Most
IDHwt, pTERTwt glioblastomas do not have EGFR amplification or À7/À10 Overall, gain/amplification of chromosome 7 was only found in 6 tumors and loss of chromosome 10 was detected in another 12 tumors. None of the tumors in this cohort harbored combined whole chromosome +7/À10. For the entire cohort, only 25 cases (35%) showed EGFR amplification and since the cases of this cohort were already pTERTwt, this suggests that three molecular features of TERT promoter mutation, EGFR amplification or combined whole chromosome +7/À10, may not molecularly cover all cases of IDHwt glioblastomas [40,41]. Interestingly, EGFR and PDGFRA amplification together covered 67% of the whole cohort and EGFR and PDGFRA amplification/mutation together covered 71% of cases successfully sequenced and the coverage would be broader.
For correlation with clinical outcomes, MGMT methylation was detected in 30/72 (41.7%) and was not associated with OS (p = 0.421) and PFS (p = 0.589) ( Figures S5A,B). CDKN2A/B homozygous deletion was associated with a worsened OS (p = 0.031, Figure 2E) and a trend for worse survival with PFS (p = 0.088, Figure 2F). Multivariate analysis including age, sex, location, resection, chemotherapy, radiotherapy, MGMT methylation, EGFR, PDGFRA, and CDKN2A/B revealed that CDKN2A/B deletion was an independent prognosticator (Table 3). They were the only genes the mutations or amplifications or losses of which were associated with OS (Tables S3 and S8).

| DISCUSSION
While pTERT mutations account for about 70%-80% of glioblastomas and are generally regarded as a late or terminal event of gliomagenesis [4,7] , whether it has a prognostic significance within glioblastomas is still uncertain. We compared our cohort with the survival of the TCGA adult glioblastoma cohort which were IDHwt, pTERTmut. No survival difference could be found between the cohorts. Similar finding of a lack of prognostic significance for pTERT mutation among IDHwt glioblastomas was also reported by Ceccarelli et al. [16]. Diplas et al. also showed no difference in survival between IDHwt, pTERTmut, and IDHwt, pTERTwt glioblastomas, although with smaller cohorts [3]. IDHwt, pTERTwt glioblastomas also seem to occur in younger patients LGm6-GBM (frequency and number of cases) p-Value (non-LGm6-GBM vs. LGm6-GBM tumors) than IDHwt, pTERTmut patients in our series, and similar finding was observed in other studies [5,6]. Importantly, in this cohort, we found that only 25 cases (35%) showed EGFR amplification and no case showed combined whole chromosome +7/À10. As these cases were already pTERTwt, so the WHO 2021-listed molecular diagnostic features of IDHwt glioblastomas, namely either TERT promoter mutation, EGFR amplification or combined whole chromosome +7/À10 cannot cover all cases of IDHwt glioblastomas in this context. It should be noted that even in Stichel and von Deimling study found the sensitivity of the three markers was only 77.8% for coverage of IDHwt glioblastomas [42]. Interestingly, in this cohort, EGFR and PDGFRA amplifications covered 67% of cases and EGFR/PDGFRA amplification or mutation together covered 71% of cases successfully sequenced, suggesting that adding PDGFRA alterations in the umbrella of molecular features of IDHwt glioblastomas may be more inclusive. Interestingly, a recent paper by Fujimoto et al. showed that PDGFRA gain/amplification was an independent predictor of poor prognosis in IDHwt, pTERTwt lower-grade gliomas [43] , highlighting the potential diagnostic value of PDGFRA alteration in IDHwt lower-grade gliomas and our results are consistent with their findings. And MGMT, a major favorable prognosticator in glioblastoma as shown by many studies [44,45] , provided no survival benefit in this cohort. This concurs with the results of another study [19].
Other than mutations at the promoter of TERT, structural rearrangement upstream of TERT locus has been associated with telomerase activity [3,46]. Diplas et al. found that other than ATRX mutation, SMARCAL1 mutation is another mechanism for ALT in gliomas [3]. For mechanism of telomere maintenance, overall in this cohort, 44 cases (61%) showed ALT, 4 cases showed ATRX mutations, 2 cases showed SMARCAL1 mutations and 17 cases (24%) showed TERT structural rearrangement, and LGm6-GBM cases predominantly showed ALT (73%). ATRX and SMARCAL1 were mutually exclusive similar to findings of Diplas et al. In 23 cases (32%) in our cohort, the mechanism of telomere maintenance was not accounted for and ATRX and SMARCAL1 mutations did not account for 88% of cases exhibiting ALT. We concede that the methods for telomere maintenance in cancer are diverse and we have not covered all possible mechanisms for telomerase activation in this study [1]. Similarly, in a smaller series, Williams et al. [6] only found ATRX mutations in 6/16 (37.5%) cases of pTERTwt glioblastomas. Interestingly, the group of "mechanism not yet found" cases in our cohort showed an enrichment of EGFR amplification (52%), this being biomarker of high-grade gliomas [47,48]. For the whole cohort, homozygous deletion of CDKN2A/B was associated with a worse OS (p = 0.031) and it was an independent prognosticator in multivariate analyses. CDKN2A/B deletion was found in all methylation groups and in both ALT and non-ALT tumors. This is interesting in that CDKN2A/B deletion is also an independent poor prognostic marker for IDH mutant lowergrade astrocytomas [31,49] and this is the only gene where mutations or CNVs showed a survival significance in this cohort.
Other than providing detailed molecular landscape for adult IDHwt, pTERTwt glioblastomas, the findings of this study also have implications for diagnostic practice where it may be impracticable or too expensive to perform methylation studies. Our findings show that the current molecular features to define molecular glioblastomas were seen only in a subset of IDHwt glioblastomas which are already pTERTwt, and that EGFR and PDGFRA amplification or mutation studies may cover most cases of IDHwt glioblastomas when the tumor is already TERTwt. Among IDHwt glioblastomas that are pTERTwt, our findings show that CDKN2A/B homozygous deletion, a test that is easily performed by FISH and is already routinely used in diagnostic laboratories for IDH mutant astrocytomas, is an independent poor prognosticator in this group of adult gliomas.

AUTHOR CONTRIBUTIONS
Study conception and design: EML and HKN performed study concept and design; EML, ZFS, KKL, NYC, HC, JYC, MFP, DTC and YM performed experiment work and data acquisition; EML, TMM, JSK, HN and HKN performed data analysis and interpretation; KKL and HKN prepared the manuscript. All authors read and approved the final paper.

FUNDING INFORMATION
This study was supported by the Health and Medical Research Fund (HMRF), the Food and Health Bureau of Hong Kong (reference number: 07180736); Children Cancer Foundation; National Natural Science Foundation of China (reference number 82072020); and, Shanghai Municipal Science and Technology Major Project, China (reference number 2018SHZDZX01).