TERT Mutations in Glioma: ESMO Biomarker Factsheet

TERT in cancer

Telomerase reverse transcriptase (TERT) is a gene located on chromosome 5p15.33, and encodes for the catalytic subunit of telomerase [1]. Telomerase is an enzyme whose mechanism of action consists on the addition of nucleotides to telomeres. In well differentiated cells from normal tissue, the activity of telomerase is relatively low, which allows cell senescence and apoptosis.

The abnormal reactivation of telomerase complex occurs in approximately 90% of human tumours, and is considered a crucial element for cancer genesis and progression [2]. The increase of TERT expression is mainly due to activating somatic point mutations that substitute a cytosine for a thymidine at position 228 (C228T) and 250 (C250T) of the TERT gene promoter (pTERT). These mutually exclusive mutations are typically heterozygous and result into an identical 11 base pair sequence (‘CCCGGAAGGGG’) which represents an E26 transformation-specific family transcription factor (ETS) binding site recognised by GABPA, a component of the multimeric transcription factor GABP [3]. Mutations of pTERT occur at high frequency in a number of malignancies, including glioma, urothelial cancer, melanoma, and thyroid cancers [1].

TERT in glioma

In glioma, pTERT mutations are commonly found in oligodendroglioma and primary glioblastoma multiforme (GBM) [4]. In a study of more than 200 specimens of gliomas, pTERT mutations occurred in 78% of oligodendrogliomas and 83% of GBMs IDH-wildtype (also called primary GBM) [5]. More recently, a larger study conducted on more than 1000 gliomas, found pTERT mutation in virtually all cases of grade II and III oligodendrogliomas (Table 1) [6]. By contrast, pTERT mutation was present in only 10% of astrocytomas and GBM IDH-mutant (also called secondary GBM) (Table 1) [6]. On this basis, assessment of pTERT mutation status represents a valuable additive tool to IDH mutation and 1p19q co-deletion in order to refine the World Health Organization (WHO) 2016 classification of gliomas [6,7].

TERT as a prognostic biomarker

The simultaneous assessment of pTERT mutation with other genetic aberrations, such as IDH mutation and 1p/19q co-deletion, may help to define the prognosis of gliomas, particularly grade II and III tumours (lower-grade gliomas, LGGs) [6,7]. In a large-scale analysis, on over 1000 cases, it was demonstrated that LGGs that were positive for all three markers, so called ‘triple positive’, had the best overall survival [7], while LGGs with only IDH mutations showed an intermediate prognosis, but still had a median survival of many years. Intriguingly, LGGs with TERT mutation only (IDH-wildtype and 1p/19q non-co-deleted) that represented 10% of all LGGs, had the worse overall prognosis, with a median survival that resembled that of GBM. Based on these findings, the presence of an isolated pTERT mutation in LGGs identifies a disease with a particularly aggressive clinical behaviour.

By contrast, in higher-grade gliomas (HGGs) such as GBM the combination of pTERT mutationwith IDH mutationand 1p/19q status did not result into a significant discrimination of the overall prognosis [7]. However, it was shown that GBMs with mutations in TERT with or without IDH mutations (2% and 74% of the total, respectively) had a similarly poor overall survival, with a prognosis that was much worse than GBMs with IDH mutation only (7% of the total), and slightly worse than GBMs negative for both pTERT and IDH mutation (17% of the total). More recently, a study suggested the possibility of detecting pTERT mutations in tumour-derived DNA from cerebro-spinal fluid (CSF) of patients with GBMs, with a good sensitivity (>90%) and a predicting potential for poor survival in presence of a high burden of pTERT-mutant alleles in the CSF tumour-derived DNA [8].

TERT as a predictive biomarker

The potential of pTERT mutation as a biomarker of sensitivity to chemotherapy for the treatment of gliomas is not yet clearly defined.

With regard to LGGs, IDH mutations seem to identify those patients who benefit from adjuvant chemotherapy given in addition to radiotherapy [9], whereas the predictive role of pTERT mutation has been poorly explored. Interestingly, pTERT mutation may have a role in predicting clinical benefit from genotoxic therapies in IDH-wildtype LGGs. A retrospective study showed that in IDH-wildtype patients, pTERT mutation identified those individuals who would experience a survival benefit from adjuvant chemotherapy or radiotherapy, while no improvement in survival was observed with the use of adjuvant treatments in IDH-wildtype/TERT-wildtype cases [10].

In HGGs, pTERT mutation seems to confer resistance to temozolomide given in combination with radiotherapy, but only in the context of MGMT-unmethylated tumours. In a retrospective analysis from Japan, primary GBM (IDH-wildtype) patients treated with concurrent temozolomide plus radiotherapy were stratified according to pTERT mutation and MGMT status. Of note, while the overall survival did not differ according to pTERT mutation within MGMT-methylated GBMs, prognosis was poorer for pTERT-mutated cases in the context of MGMT-unmethylated group [11]. These results were corroborated by a subsequent analysis of an US cohort of primary GBMs who were similarly treated with concurrent temozolomide and radiotherapy [12]. Although both studies are biased owing to their retrospective nature, they suggest that the presence of pTERT mutation may hold predictive value based on MGMT gene promoter methylation status.

TERT testing recommendations

Two different technical approaches can be exploited to identify pTERT mutations, namely Sanger sequencing or pyrosequencing. The latter enables the quantification of the genomic variant as a ‘pyrogram’, thus allowing the detection of a small fraction of positive cells in samples with low tumour cell content. However, pyrosequencing is preferred when samples undergo large genomic screenings aimed to offer the opportunity of assessing a large set of genomic targets which can be relevant for clinicians. In all other cases, Sanger sequencing is the most largely applied technique. The experiment is conducted by using genomic DNA and a specific pair of primers to cover the whole promoter region and identify all mutation variants [13,14].In order to identify all pTERT variations, the forward and reverse primers should be chosen to map at about 220 bp upstream and 60 bp downstream to the ATG codon. To separate the PCR products, they are run through agarose electrophoretic gel matrix, bands are manually cut, recovered, and sequenced by Sanger’s method. The data obtained from this technique is ‘qualitative’, which means presence or absence of mutations with no information on variant allele frequency.

Ensuring quality and timely testing results

Nowadays,pTERT mutations analysis is limited to DNA-based methods, namely sequencing or pyrosequencing, the latter in the context of next generation sequencing studies including a number of genomic targets, and no external quality control to assess their analytic performance has been yet reported. General advices, for the most routinely applied Sanger sequencing method, are to use normal and mutated control samples to set up primers and PCR conditions in order to establish the specificity and sensitivity of experimental conditions. Moreover, to rule out false positive results and ensure the reproducibility of the analysis, cases with sequence variations should be studied in duplicated. As previously indicated, the sensitivity of molecular techniques is assured by a careful selection of neoplastic area to avoid admixture of mutated DNA from neoplastic cells and non-mutated DNA from normal cells [14].

Patient selection

Although the assessment of pTERT mutation status is not ready for prime time in terms of patients stratification according to the WHO 2016 classification of gliomas, its determination has a potential in order to assist histological diagnosis. In fact, detecting a pTERT mutation in LGGs in association with IDH mutation and 1p/19q co-deletion may serve as a diagnostic surrogate for oligodendroglial lineage.

With regard to prognosis of LGGs, pTERT mutation in the absence of either IDH mutation and 1p/19q co-deletion clearly identifies a group of tumours with an aggressive clinical course [7]. In this context, the assessment of pTERT mutation status could provide relevant information on which LGGs should be treated aggressively with early adjuvant therapies and/or require close follow-up. On the other hand, as for HGGs, accumulating evidence suggests that detecting a pTERT mutation in primary GBMs could serve as an indicator of poor survival in primary GBMs [7,8].

References

1. Liu T, Yuan X, Xu D. Cancer-specific telomerase reverse transcriptase (TERT) promoter mutations: biological and clinical implications. Genes (Basel)  2016; 7(7):38.
2. Heidenreich B, Rachakonda PS, Hemminki K, Kumar R. TERT promoter mutations in cancer development. Curr Opin Genet Dev 2014; 24:30-7.
3. Bell RJ, Rube HT, Kreig A, et al. Cancer. The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer. Science 2015; 348(6238):1036-9. 
4. Yuan Y, Qi C, Maling G, et al. TERT mutation in glioma: frequency, prognosis and risk. J Clin Neurosci 2016; 26:57-62.
5. Killela PJ, Reitman ZJ, Jiao Y, et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proc Natl Acad Sci U S A 2013; 110(15):6021-6. 
6. Pekmezci M, Rice T, Molinaro AM, et al. Adult infiltrating gliomas with WHO 2016 integrated diagnosis: additional prognostic roles of ATRX and TERT. Acta Neuropathol 2017; 133(6):1001-6.
7. Eckel-Passow JE, Lachance DH, Molinaro AM, et al. Glioma groups based on 1p/19q, IDH, and TERT promoter mutations in tumors. N Engl J Med 2015; 372(26):2499-508.
8. Juratli TA, Stasik S, Zolal A, et al. TERT promoter mutation detection in cell-free tumor-derived DNA in patients with IDH wild-type glioblastomas: a pilot prospective study. Clin Cancer Res 2018;24(21):5282-91.
9. Cairncross JG, Wang M, Jenkins RB, et al. Benefit from procarbazine, lomustine, and vincristine in oligodendroglial tumors is associated with mutation of IDH. J Clin Oncol 2014; 32(8):783-90.
10. Zhang ZY, Chan AK, Ding XJ, et al. TERT promoter mutation contribute to IDH mutation in predicting differential responses to adjuvant therapies in WHO grade II and III diffuse gliomas. Oncotarget 2015; 6(28):24871-83.
11. Arita H, Yamasaki K, Matsushita Y, et al. A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas. Acta Neuropathol Commun 2016; 4:79. 
12. Nguyen HN, Lie A, Li T, et al. Human TERT promoter mutation enables survival advantage from MGMT promoter methylation in IDH1 wild-type primary glioblastoma treated by standard chemoradiotherapy. Neuro Oncol 2017; 19(3):394-404.
13. Yang P, Cai J, Yan W, et al. Classification based on mutations of TERT promoter and IDH characterizes subtypes in grade II/III gliomas. Neuro Oncol 2016; 18(8):1099-108.
14. Bieńkowski M, Wöhrer A, Moser P, et al. Molecular diagnostic testing of diffuse gliomas in the real-life setting: a practical approach. Clin Neuropathol 2018; 37(4):166-77.