TERT Alterations Predict Tumor Progression in De Novo High-Grade Meningiomas Following Adjuvant Radiotherapy

Background Adjuvant radiotherapy (RT) is one of the most commonly used treatments for de novo high-grade meningiomas (HGMs) after surgery, but genetic determinants of clinical benefit are poorly characterized. Objective We describe efforts to integrate clinical genomics to discover predictive biomarkers that would inform adjuvant treatment decisions in de novo HGMs. Methods We undertook a retrospective analysis of 37 patients with de novo HGMs following RT. Clinical hybrid capture-based sequencing assay covering 184 genes was performed in all cases. Associations between tumor clinical/genomic characteristics and RT response were assessed. Overall survival (OS) and progression-free survival (PFS) curves were plotted using the Kaplan–Meier method. Results Among the 172 HGMs from a single institution, 42 cases (37 WHO grade 2 meningiomas and five WHO grade 3 meningiomas) were identified as de novo HGMs following RT. Only TERT mutations [62.5% C228T; 25% C250T; 12.5% copy number amplification (CN amp.)] were significantly associated with tumor progression after postoperative RT (adjusted p = 0.003). Potential different somatic interactions between TERT and other tested genes were not identified. Furthermore, TERT alterations (TERT-alt) were the predictor of tumor progression (Fisher’s exact tests, p = 0.003) and were associated with decreased PFS (log-rank test, p = 0.0114) in de novo HGMs after RT. Conclusion Our findings suggest that TERT-alt is associated with tumor progression and poor outcome of newly diagnosed HGM patients after postoperative RT.

Patients with WHO grade 1 meningiomas are traditionally managed in follow-up with surveillance imaging (9). However, a standardized treatment approach to HGMs after resection has not been established (4). The benefit to survival outcomes of HGMs with adjuvant radiotherapy (RT) post-surgical resection remains unclear. Retrospective series on adjuvant RT after gross total resection led to differing results (4,10,11).
Meningiomas have a diverse genetic background that varies with biologic behavior (12). Alterations in the tumor suppressor gene NF2 were the first discovered genetic etiology of meningiomas (13,14). In NF2 wild-type meningiomas, mutations in TRAF7, KLF4, AKT1, and SMO were noted (15)(16)(17). In addition, several mutations have been described with potential prognostic implications in HGMs (12,14,18). Data published recently have also shown that activating TERT promoter mutations, frequent inactivation of BAP1, deletions of CDKN2A/ B, and mutations in DMD are frequent in meningiomas with malignant histological progression (18)(19)(20)(21). These data suggest that convergent gene-expression programs may underlie HGMs, which could be leveraged to develop prognostic biomarkers.
Our previous work found that patients with de novo anaplastic meningiomas benefit from adjuvant RT after surgery (5,22). However, the molecular factors associated with RT efficacy in de novo HGMs are largely unknown. In the present study, we describe efforts to integrate clinical genomics of 37 cases from 173 HGMs to address this issue.

Patient Selection
Patients were identified for study through a review of the clinical records of the Department of Neurosurgery, Huashan Hospital of Fudan University, Shanghai, China. A total of 172 HGMs (Supplementary Table S1) were included following study approval by the Human Subjects Institutional Review Board at Huashan Hospital, Fudan University (KY-2017-09). Clinical characteristics including age, gender, tumor location, extent of surgical resection, and outcome data were collected. In general, adjuvant radiation was recommended to both atypical and anaplastic meningioma patients, regardless of gross total resection (GTR; Simpson grades I-III) or subtotal resection (STR; Simpson grades IV-V). And the final decision was made based on the negotiation with the relatives of patients. The details of postoperative RT were described in our previous work (5). Tumor pathological subtypes were reconfirmed by at least two experienced neuropathologists. Follow-up was conducted routinely according to the guidelines of Huashan Neurosurgical Center. Written informed consent was obtained from all patients involved in our study.
A total of 172 patients with a confirmed diagnosis of HGM who met inclusion criteria (141 WHO grade 2 meningiomas and 31 WHO grade 3 meningiomas; Supplementary Table S1). Out of 172 cases, 87 cases received RT after surgery. Among the 87 patients, 42 (48.3%) were de novo meningiomas, while the remaining 45 patients presented with recurrent meningioma following prior surgical resection. And finally, 37 de novo meningioma samples with adequate quality of DNA concentration were included for further next-generation sequencing.

Sanger Sequencing
The TERT promoter mutations were evaluated using Sanger sequencing. Genomic DNA was obtained from FFPE using the HiPure FFPE DNA Kit (Magen, D3126-03) following polymerase chain reaction-based amplification of the target region (forward primer: GGATTCGCGGGC ACAGAC; reverse primer: CAGCGCTGCCTGAAA CTC; details on PCR conditions are available upon request).

Statistical Tests
The specific details of statistical tests are included in the figure legends. A two-tailed Fisher's exact test was used to calculate statistical significance between different groups using a c 2 2 × 2 table. Categorical variables were compared with the Fisher's exact tests, and continuous variables with the independentsamples Student's t-test (data with normal distribution) or Mann-Whitney U-test (data with skewed distribution). Continuous data were expressed as the mean ± standard deviation (SD). Overall survival (OS) and progression-free survival (PFS) curves were plotted using the Kaplan-Meier method. Statistical analysis was performed using Statistical Package for Social Sciences (SPSS, Version 20.0, Chicago, IL, USA). Data were considered to be significant when p < 0.05.

TERT Mutations Predict Tumor Progression of De Novo High-Grade Meningiomas Following Radiotherapy
Strikingly, only TERT mutations were significantly associated with tumor progression (n = 8, adjusted p = 0.031), and all these mutations were present in tumors that progressed after RT ( Figure 3A). Of the TERT mutant cases, 87.5% (7/8) presented with TERT promoter mutations (62.5% C228T variant and 25% C250T variant; Supplementary Figure S1), and 12.5% (1/8) harbored copy number amplification (CN amp.). As many tumor driver genes are co-occurring or show strong exclusiveness in their mutation pattern, we next explored the potential different somatic interactions in the cohort. None of the gene mutations   Figure S2). Thus, TERT mutations appear to be the dominant cause of tumor progression among mutations in this cohort. Furthermore, we found that TERT mutation status (p = 0.003), as well as extent of resection (EOR) (p < 0.001), was significantly associated with tumor progression by Fisher's exact tests ( Table 1). Neither tumor location (p = 1.00) nor WHO grade (p = 1.00) predicted tumor progression after postoperative  RT. After adjustment for multiple comparisons, these associations were not significant. Importantly, TERT mutation cases were more likely to have unfavorable time to recurrence (TTR) over the entire cohort, with a mean observation time of 47.6 months ( Figure 3B). However, no significant differences in the average time to recurrence between TERT mutant and wildtype cases were observed (Wilcoxon rank sum test with continuity correction W = 491, p = 0.0864). Meningioma with TERT alterations, regardless of WHO grades or pathological subtypes, had a highly significant risk of recurrence (26). To exclude the disruption to RT efficacy might be caused by TERT-related malignant biological behavior, we thus performed analysis on newly diagnosed HGMs depending on TERT alterations only in progression group. Of the 19 cases in the progression group, mitotic index (ki-67%) depending on TERT alterations did not show any significantly difference (unpaired t-test, p = 0.051; Supplementary Figure S3A).
Additionally, TERT alterations of de novo HGMs had no predictive effect on tumor recurrence in progression group following postoperative RT (p = 0.074 with log-rank test; Supplementary Figure S3B).

TERT Mutations Were Associated With Decreased Progression-Free Survival and Overall Survival in De Novo High-Grade Meningiomas After Radiotherapy
With these findings, we next analyzed the effect of TERT mutation status on PFS in patients with de novo HGMs after RT. The primary endpoint of PFS, defined as time from surgery to date of progression, was assessed on the basis of progression of meningioma after initial surgery on imaging follow-up. The median PFS of 75 months (range: 7-109 months) was observed for the entire cohort. Subgroup analysis revealed a median PFS of 25 months (range: 10-79 months) in TERT mutant group and 77 months (range: 7-109 months) in TERT wild-type group. TERT mutants were significantly associated with decreased PFS in de novo HGM cases that underwent postoperative RT (p = 0.0114 with log-rank test; Figure 4A).
The relationship between TERT mutations and OS was analyzed using the log-rank test ( Figure 4B). The median OS of 84 months (range: 13-123 months) was observed for the entire cohort. Subgroup analysis revealed a median OS of 66 months (range: 13-123 months) in TERT mutant group and 85 months (range: 24-109 months) in TERT wild-type group. Patients with de novo HGMs after RT harboring TERT mutations had worse OS (p = 0.0562; Figure 4B).

DISCUSSION
In this retrospective study, we investigated genetic predictors that might inform the potential progressive risk in de novo HGMs after postoperative RT. We found TERT-alt in HGMs to be a significant predictor of tumor progression compared to TERT wild-type cases. Although a merely descriptive finding, our  data have potential implications for the clinical management of patients with TERT-alt de novo HGMs. HGMs are rare but aggressive tumors with considerably poorer prognosis than lower-grade meningiomas. Adjuvant RT is the only nonsurgical standard of care treatment option for these tumors (9). However, radiotherapeutic options for meningioma are diverse, and there are no randomized trials to identify individuals who are more likely to benefit from adjuvant RT. Indeed, until recently, the evidence supporting postoperative RT for meningioma, especially HGMs, was largely limited. Moreover, no molecular prognostic markers have yet been established for new diagnosed HGMs following RT. To our knowledge, this study is the first to investigate predictive biomarkers that would inform tumor progression in de novo HGMs after postoperative RT.
Among the 184 sequenced genes, only TERT alterations were significantly associated with tumor progression (n = 8, adjusted p = 0.031), and all these mutations were present in tumors that progressed after RT. TERT-alt comprise, but are not limited to, promoter mutations, gene translocations, and DNA amplifications (28). We found 87.5% (7/8) of the TERT mutant cases presented with TERT promoter mutations (62.5% C228T variant and 25% C250T variant). As reported, the most common alterations occur in specific "hotspots" of the promoter (TERTp) region known as C228T and C250T (29). These C>T transition mutations lead to maintenance of the telomere length, as binding of E-twenty six (ETS)-transcription factors is involved in the upregulation of TERT expression (28,30).
TERT alterations, specifically TERT promoter mutations, have been identified in a subset of HGMs with progression from low-grade meningioma (31)(32)(33)(34). In the present study, we found that TERT-alt was associated with decreased PFS and OS in de novo HGMs after RT. Besides, our data have shown decreased time to progression among TERT-alt de novo HGMs as well. TERT gene is transcriptionally inactive in most nonneoplastic cells (28). TERT gene alterations (TERT-alt) may enforce cell immortalization by counteracting telomere shortening, thus promoting growth (28). Several studies have provided evidence that TERT-alt mutations are associated with rapid recurrence and malignant progression in meningioma (26,31,35). In addition, TERTp meningiomas have been found to have a worse PFS and OS, though not many cases have been reported (18). Activating TERT gene mutations in the upstream promoter allows overexpression of this enzyme and is responsible for immortalization of tumor cells in many cancers (12). Thus, we have excluded that the disruption to RT efficacy might be caused by TERT-related malignant biological behavior in this study. As results, the TERT-alt status and progressive variables did not show any relevance in progression group.
The Simpson grade of EOR has long been used to guide clinical expectations after resection of meningiomas (9); our results support the relevance of EOR in recurrence of HGMs as well (p < 0.001). The literature widely recognizes the role that EOR plays in determining HGM prognosis (36,37). However, Cox regression analysis failed to identify any factor with significant association with the progression of de novo HGMs following RT. Thus, a larger cohort or multicenter clinical trial is needed to investigate the effect of RT in this subgroup.
In summary, our data identified TERT alterations, especially TERTp mutation, are associated with tumor progression and poor outcome of newly diagnosed HGM patients after postoperative RT. Several limitations of this study warrant consideration. Firstly, our findings on a discovery series were not substantiated by any independent validation series due to the limited available samples. Prospective studies are clearly needed to validate TERT-alt status of radiation response in de novo HGMs. Another important limitation in this study is its observational nature, which could have led to selection bias. It would be useful to repeat these analyses in cohorts from other institutions in the future.

CONCLUSION
In summary, examining a cohort of de novo HGMs following adjuvant RT, we find TERT alteration to be strongly associated with tumor progression and poor outcome of HGM patients included in this study.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI [accession: PRJNA753598].

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Human Subjects Institutional Review Board at Huashan Hospital, Fudan University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
JD, LH, and YG, study design. JD, SS, JC, DW, and QX, clinical data. JD, article preparation. HaC and HoC, tumor pathological subtypes reconfirmed and Sanger sequencing. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by grants from the National Key R&D Program of China (2018YFC1312600 and 2018YFC1312604 to YG) and the National Natural Science Foundation of China (82072788 and 81772674 to YG).
Supplementary Figure S2 | Heatmap of mutually exclusive or co-occurring set of genes in the mutation pattern of de novo HGMs following RT. Pairwise Fisher's exact tests were performed to detect significant pairs of genes, mutually exclusive or co-occurring set of genes which colored by brown or green can be detected using the somaticInteractions function in R/Bioconductor package 'maftools'.
Supplementary Figure S3 | The TERT-alt status and progressive variables did not show any relevancy in progression group.