High Incidence of Activating TERT Promoter Mutations in Meningiomas Undergoing Malignant Progression

Abstract

Meningiomas are common central nervous system tumors. The World Health Organization (WHO) defines three grades, predictive of the risk of recurrence. These tumors can relapse frequently and sometimes undergo malignant transformation. Maintenance of telomere length is a key process in malignant progression, and mutations in TERT promoter have recently been identified in various types of cancer. We sequenced the TERT promoter in 85 meningiomas from 73 patients. We found a high incidence of TERT promoter mutations in patients with meningiomas undergoing malignant histological progression (28%, n = 5/18 patients). In this subset of patients with histological progression, TERT promoter mutations were found in both the lowest and the highest grade tumors, and in both NF 2‐mutated and nonmutated samples. In contrast, one mutation was identified in 35 meningiomas without recurrence or progression, belonging to various histological grades. This sample was an aggressive meningioma in a patient who died shortly after surgery. Interestingly, tumors showing relapse without histological progression were not mutated for TERT promoter (n = 20). Finally, TERT promoter mutations were associated with a marked increase in TERT expression. Thus, TERT promoter mutations are pivotal genetic alterations involved in malignant progression of meningiomas and could be used as a biomarker to identify meningiomas at risk of malignant transformation.

Introduction

Meningiomas are the most frequent intracranial tumors and account for more than 35% of tumors 29. They develop from the meninges covering the brain and spinal cord. Although 70%–80% are slow‐growing benign WHO (World Health Organization) grade I tumors, recurrence is frequently observed, and a subset of these tumors can even undergo a malignant progression 34. These patients are difficult to manage and require new biomarkers and innovative treatments in order to improve their clinical care. Mutations in the neurofibromatosis 2 (NF2) tumor suppressor gene and loss of chromosome 22q are the most common genetic alterations involved in the initiation of meningioma tumorigenesis. Recently, new genes involved in meningioma tumorigenesis have been identified by whole genome or whole exome sequencing, including TRAF7, KLF4 (especially in secretory meningiomas), AKT1, SMO 5, 10, 33, 37, and germline mutations in SMARCB1, SMARCE1 and SUFU 1, 26, 40. Mutations in these genes seem to be mutually exclusive with NF2, and seem to be involved in the initiation of tumorigenesis. In contrast, knowledge about mechanisms underlying malignant progression remains limited. We and others have shown that progression to grade III tumors is associated with CDKN2A/B loss and accumulation of chromosome gains and losses 4, 14, 30, 42, and it was confirmed recently in a genetically modified mouse model that Nf2 and Cdkn2ab inactivation cooperate to promote meningioma progression 31. Other alterations are more frequently encountered in higher grade human tumors, but genetic events responsible for activations or losses of expression remain unclear 34.

One of these alterations is telomerase activation. Telomerase activation has been demonstrated by telomere repeat amplification protocol (TRAP) assay or quantitative polymerase chain reaction (PCR) of TERT in about 10% of grade I meningiomas, 50% of grade II and around 95% of grade III meningiomas 3, 7, 8, 9, 11, 12, 13, 15, 18, 19, 22, 23, 24, 25, 32, 36, 38, 39. Nevertheless, mechanisms underlying telomerase activation remain unknown in meningiomas.

Recently, mutations in the promoter of the telomerase reverse transcriptase (TERT) have been identified in melanomas with frequencies up to 85% of tumor and cell line samples 16, 17. These mutations create a DNA consensus sequence (CCGGAA) predicted to bind ETS/TCF transcription factor and to increase TERT transcription. Several other types of cancer have been screened and displayed recurrent somatic TERT promoter mutation, including gliomas, mesotheliomas, and bladder and hepatocellular cancers 2, 20, 27, 28, 41. In liver carcinogenesis, although no mutation has been identified in benign hepatocellular adenomas, somatic TERT promoter mutation was identified as a key genetic event involved in the malignant transformation of hepatocellular adenomas into carcinomas 27.

Whereas Killela and collaborators found no mutations in 20 unselected meningioma samples 20, we hypothesized that TERT promoter mutations could be involved in meningioma malignant progression. Thus, we screened a large series of meningiomas including 35 patients without tumor relapse and 38 patients with tumor relapse, and we identified frequent TERT promoter mutations almost exclusively in meningiomas showing recurrence associated with an increased histological grade.

Materials and Methods

Three groups of meningiomas, totaling 85 samples in 73 patients, were selected under appropriate informed consent, and all DNA and RNA were extracted from frozen tissues. The first group was composed of 30 tumor samples in 18 patients who have been operated on at least twice for tumor relapse, with proven histological progression from grade I to II (3 patients), II to III (11 patients) or I to III (4 patients). Mean time between grade I and II samples was 61 ± 64 months, and 44 ± 26 months between grade II and III (Supporting Information Table S1). The second group was composed of 20 samples from 20 patients who have been operated on at least twice for tumor relapse with identical histological grading (recurrence without histological progression: 4, 12 and 4 patients with grade I, II, and III, respectively). Mean time between the two surgeries was 73 ± 52 months. After an additional follow‐up of 49 ± 48 months, nine patients had no meningioma on magnetic resonance imaging (MRI), five had stable remnants and six slow‐growing recurrences (Supporting Information Table S2). The third group was composed of 35 single surgery meningioma samples in 35 patients, including 10 NF2 patients (10, 22 and 3 grade I, II and III meningiomas, respectively). The mean radiological follow‐up after surgery was 60 ± 40 months. At the end of follow‐up, 23 patients had no meningioma on MRI, 10 had stable remnants and 2 died before radiological evaluation (Supporting Information Table S3). When multiple samples from the same patient were analyzed, samples were identified by a number corresponding to the patient, and by letters A, B or C, meaning first, second or third sample, respectively. Procedures for case selection, DNA and RNA extraction, NF2 gene mutation status assignment and Affymetrix SNP 500K, SNP 5.0 and SNP 6.0 array analysis were performed as described elsewhere 14. Single nucleotide polymorphism (SNP) array was performed in 72 samples.

The TERT promoter including bases covering −70 to −220 nucleotides from TERT ATG was PCR amplified and sequenced by Sanger technic as previously described 27. Quantitative reverse transcription (RT)‐PCR was performed in 56 tumor RNAs with good quality as previously described 27. Expression was assessed in duplicate using TaqMan (Applied Biosystems, Foster City, CA, USA) gene expression assay (TERT Hs00972656_m1). The relative amount of RNA was calculated with the 2‐ΔΔCT method. Gene expression was normalized with the RNA ribosomal 18S, and the level of expression of the tumor sample was compared with the median level of the gene expression in the samples and expressed as an n‐fold ratio. Statistical analysis was performed using GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA), using appropriate tests. Data are presented as number (percent) or means ± standard deviation (SD) as appropriate for the type of data.

Results

TERT promoter mutation is an early genetic event specific of meningioma progression

Among the 85 screened meningiomas, TERT promoter mutations were identified in 9 samples (9/85, 11%), from 6 patients (6/73, 8%) (Table 1). These mutations were typical −124G > A (g.1,295,228G > A, 7/9 samples) and −146G > A (g.1,295,250G > A, 2/9 samples), as described in other types of tumor. Mutations in these two hot spots were mutually exclusive. Although normal tissues were not available to confirm the somatic status of the mutations identified, we have assumed the somatic status because all published studies have systematically confirmed that g.1,295,228G > A (−124G > A from the ATG start site) and g.1,295,250G > A (−146G > A from the ATG start site) mutations were somatic. Interestingly, 8/9 mutated samples were from 5 progressing meningioma patients: 1 grade I to II, 2 grade II to III and 2 grade I to III (8/30 progressing meningioma samples: 27%, 5/18 patients: 28%). In contrast, no TERT promoter mutation was identified in 20 patients with recurring meningiomas without histological progression (P = 0.017, Fisher's exact test). Similarly, only one mutation was identified among single surgery samples from various pathological grades (35 patients). This mutated sample (#252) was an aggressive grade II meningioma in a patient who died shortly after surgery, before any histological progression could occur. When the low and higher grade samples were available for a single patient, the identical TERT promoter mutation has always been identified in the two samples (three patients). Among progressing samples, NF2 inactivating mutations were present in 76% of the cases. TERT mutations were identified in both NF2‐mutated and nonmutated samples (n = 7 and 2, respectively). Clinical and molecular features of TERT promoter‐mutated and nonmutated samples were similar, except for a higher rate of malignant progression (Table 2): malignant progression occurred in 5/6 patients (88%) with TERT promoter mutation vs. 13/67 patients (19%) without TERT promoter mutation (P = 0.003, Fisher's exact test).

Table 1.

TERT promoter mutations and NF 2 status of progressing meningioma samples. Abbreviations: SNP = single nucleotide polymorphism; WHO = World Health Organization

Patient ID	Sample ID	Sample WHO grade	Patient WHO grade	TERT promoter mutation	Biallelic NF2 inactivation	NF2 mutation	22q loss (SNP array)
89	89.A	II	I to III	g.1,295,228G > A, −124G > A	Yes	c.955C > T, p.Gln319X	Yes
	89.B	III		g.1,295,228G > A, −124G > A	No	None	No
91	91.A	I	I to II	g.1,295,228G > A, −124G > A	Yes	c.743del C, p.Asn248fs	Yes
	91.B	II		g.1,295,228G > A, −124G > A	Yes	c.743del C, p.Asn248fs	Yes
119	119.A	II	I to III	g.1,295,228G > A, −124G > A	Yes	c.1263G > T, p.Glu422X	Yes
207	207.A	II	II to III	g.1,295,250G > A, −146G > A	Yes	c.203_204insA, p.Ileu68fs	Yes
	207.B	III		g.1,295,250G > A, −146G > A	Yes	c.203_204insA, p.Ileu68fs	Yes
213	213.A	II	II to III	g.1,295,228G > A, −124G > A	No	None	No
252	252	II	II*	g.1,295,228G > A, −124G > A	Yes	c.599 + 1G > T	Yes

*Immediate postoperative death.

Table 2.

Comparison between TERT‐mutated and nonmutated meningiomas. Abbreviation: WHO = World Health Organization

	TERT promoter‐mutated patients n = 6	TERT promoter nonmutated patients n = 67	Statistical test	P
Progression (yes/no)	5/1 (83%)	13/54 (19%)	Fisher's exact	0.0029
Recurrence (yes/no)	5/1 (83%)	33/34 (49%)	Fisher's exact	0.2
Mean age at surgery (years)	55 ± 14	51 ± 17	Mann–Whitney	0.6
Gender (female/male)	4/2 (67%)	36/31 (54%)	Fisher's exact	0.68
Localization (skull base/nonskull base)	1/5 (17%)	15/50 (23%)	Fisher's exact	1.0
22q loss (yes/no)	5/1 (83%)	48/12 (80%)	Fisher's exact	1.0
NF2 mutated	5/1 (83%)	38/19 (67%)	Fisher's exact	0.65
WHO grade (I/II/III)	1/5/0 (17/83/0%)	18/39/10 (27/58/15%)	χ2	0.2
Brain invasion (yes/ no/no visible brain)	1/2/3 (17/33/50%)	15/16/36 (22/24/54%)	χ2	0.9
Mitosis (mean ± SD)	5.0 ± 4.7	6.0 ± 10	Mann–Whitney	0.97
Mean fractional allele loss 14	0.29 ± 0.07	0.21 ± 0.13	Mann–Whitney	0.05

Increased TERT expression in meningiomas with TERT promoter mutations

We assessed TERT expression by quantitative RT‐PCR in 54 samples: 12 progressing patients (23 samples), 16 single surgery patients, 15 patients with the same grade recurrence (15 samples). As expected, TERT promoter‐mutated samples displayed a higher TERT expression than nonmutated samples (mean fold‐change = 65 ± 69, n = 9, vs. mean fold‐change = 3.6 ± 8.5, n = 45; Mann–Whitney, P = 0.0001; Figure 1). TERT expression was higher in grade II (mean 19.7 ± 49, n = 27) and grade III (mean 16.1 ± 19, n = 12) meningioma samples than in grade I samples (mean 1.7 ± 3.3, n = 15, Kruskal–Wallis, P = 0.008) (Figure 2). A higher TERT expression was found in progressing samples than in single surgery or same grade recurrent meningiomas (Figure 3). In progressing samples, TERT expression rose with increasing grade (Wilcoxon matched pairs test, P = 0.047). Overall, 15/23 (65%) progressing samples harbored an increased TERT expression (fold‐change > 2). Interestingly, 7/15 (47%) samples with an increased TERT expression did not exhibit TERT promoter mutation, suggesting alternative mechanisms involved in telomerase reactivation and telomere maintenance. We looked for TERT locus amplification as an alternative mechanism for telomerase reactivation, but no amplification was identified on SNP array analysis in 78 samples.

Figure 1.

TERT  expression is increased in  TERT ‐mutated meningioma samples. TERT‐mutated samples (red triangles) showed higher mean level of TERT expression than nonmutated samples (black dots) (Mann–Whitney P = 0.0001). Error bars represent SEM.

Figure 2.

TERT  expression is increased in high‐grade meningioma samples. Mean level of expression of tumor samples in grade I, II and III meningiomas. (Kruskal–Wallis P = 0.008, Dunn's multiples comparison test). Error bars represent SEM. Red triangles stand for TERT‐mutated samples, and black dots for nonmutated samples.

Figure 3.

Higher  TERT  expression in meningioma samples undergoing histological progression compared with single surgery samples and recurrent meningiomas without histological progression ( K ruskal–W allis P = 0.008). TERT promoter mutations (red triangles) are mostly identified in progressing samples. Of note, the only TERT‐mutated sample in the single surgery group is a grade II meningioma in a patient (#252) that died shortly after surgery of an extensive meningioma. Error bars represent SEM.

Discussion

In this study, we identified TERT promoter mutations in 28% of patients with progressing meningiomas, the mutation being present from the lower grade sample. In addition, our data suggest that the mutation of TERT promoter, increasing strongly telomerase expression, is a major event driving meningioma histological progression. In a previous study, we have demonstrated the key role of TERT promoter mutations in progression from hepatocellular adenomas to hepatocellular carcinomas in association with CTNNB1 mutations 27. Together with this present study, it strongly highlights the role of TERT promoter mutations in the multistep process of malignant transformation in humans. In line with this findings, a recent publication by Koelsche et al showed that 0/91 WHO grade I, 2/49 (4%) grade II and 6/37 (16%) grade III meningiomas harbored TERT promoter mutations 21. These mutated samples could correspond to meningiomas that are the consequence of a histological progression, and thus classified mainly as grade II or grade III.

TERT promoter mutations were found in both NF2‐mutated and non‐mutated samples, suggesting a major role for TERT in histological progression regardless of the NF2 status of meningioma. We have previously shown that histological progression of NF2‐mutated meningiomas is associated with chromosomal instability, which is readily seen in lower grade tumors 14. Similarly, TERT promoter mutations are detected in low‐grade tumors. This suggests that TERT promoter mutations could be an early event in the genesis of meningiomas prone to malignant progression, allowing survival of cells harboring chromosomal imbalances. According to this hypothesis, the progression to grade III would be definite once strong additional mechanisms occur, such as CDKN2A/B loss 4, 14, 30, 42.

We also demonstrated that TERT promoter mutations lead to an increased telomerase transcription as mutated samples exhibited an increase TERT transcription compared with nonmutated tumors. However, 65% of meningiomas with malignant progression have telomerase reactivation, but 47% did not harbored mutations in the TERT promoter. Thus, additional mechanisms leading to telomerase re‐expression remain to be investigated in meningiomas.

Moreover, some patients showed histological progression without an increase in TERT expression. This could suggest that other mechanisms such as ALT (alternative lengthening of telomere) could be involved in telomere maintenance in meningiomas, similar to what is observed in glioma: In a recent paper including 25 glioblastomas studied by whole genome sequencing, TERT promoter mutations and ATRX mutations were present in 100% of samples and were mutually exclusive 6.

In clinical practice, our data suggest that identification of TERT promoter mutations in phenotypically benign meningiomas could be a predictive biomarker of high risk of progression to higher histological grade. After validation in a larger, prospective series, identification of TERT mutations could lead to clinical management of patients, for example, supporting a more aggressive surgical strategy including proactive surgery of remnants, or adjuvant radiotherapy. Moreover, this could open up a new window of opportunities as therapeutics targeting telomerase are currently under development 35, and no drug has established efficiency against meningiomas.

In conclusion, our findings demonstrate a major role for TERT promoter mutations in histological progression of meningiomas.

Supporting information

Table S1. Clinical and molecular features of progressing meningioma samples.

Table S2. Clinical and molecular features of same grade recurrent meningioma samples.

Table S3. Clinical and molecular features of single surgery meningioma samples.