TERT promoter mutations in thyroid cancer

Abstract

TERT promoter mutations (TERT-p mutations) have been found in many types of cancer and have emerged to play critical roles in tumor progression. The mutations upregulate TERT transcription, and TERT not only elongates telomeres and confers unlimited proliferative capacity on tumor cells, but is also involved in tumor progression and aggressiveness. In differentiated thyroid carcinoma, TERT-p mutations are associated with a number of high-risk clinicopathological aggressiveness and worse prognosis, making it the best molecular marker to predict tumor aggressiveness so far. This review summarizes recent relevant findings regarding TERT-p mutations and their functional/mechanistic aspects.

TERT Promoter Mutations in Cancer

Telomerase Reverse Transcriptase (TERT) is a catalytic subunit of telomerase which maintains telomere structures at DNA ends and is not expressed in most human somatic cells, which is thought to be one of the mechanisms that prevents cancer in humans. In the vast majority of cancers, however, increased expression of the TERT gene is achieved by several mechanisms, including mutations in the core promoter region of the TERT gene (TERT-p mutations), thereby allowing cell immortalization which is a hallmark of cancer cells.

The first report of TERT-p mutations in cancer was published in 2013 [1, 2]. Whole genome sequencing analysis of malignant melanoma tissues revealed two highly recurrent somatic mutations in the TERT promoter: mutually exclusive heterozygous C > T mutations at –124 and –146 bp upstream from the initiation codon. They are also referred to as chr5:1,295,228 C > T (C228T) and chr5:1,295,250 C > T (C250T), respectively, based on the hg19 coordinate, but the latest human genome reference is GRCh38/hg38. TERT-p mutations have also been discovered in various other types of malignancies including bladder cancer, renal pelvic cancer, hepatocellular carcinoma, glioblastoma, and thyroid carcinoma. On the other hand, they are rarely detected in hematological malignancy, prostate cancer, gastrointestinal cancer, breast cancer, and lung cancer. Both mutations create binding sites for ETS family transcription factors and upregulate the transcription of the TERT gene.

Regarding the difference between the –124 and –146 mutations, several papers have reported that the transcriptional activity of –124 was higher than –146 in reporter assays [3-7], while others demonstrated no difference [1, 8]. As enhancers strongly regulate transcription of the human TERT gene, reporter assays using only the promoter region may not be able to reach a conclusion [9]. In studies comparing mRNA expression in clinical specimens, the results were also inconclusive [8, 10]. In melanomas, only the –124 mutation, but not –146, was associated with TERT mRNA upregulation [11]. There is also a report indicating that the –146 mutation, but not –124, requires not only ETS transcription factors but also NF-κB p52 for its transcriptional enhancement [12]. In general, the –124 mutation seems to be the more important of the two, but this too is still inconclusive and could differ depending on the type of cancer.

Interestingly, TERT-p mutations were scarcely detected in cancers arising from tissues that are always self-renewing (e.g., myeloid leukemia, breast cancer, and prostate cancer), while tumors arising from cells with low rates of self-renewal were found to have higher prevalence of TERT-p mutations (e.g., melanoma, hepatocellular carcinoma, urothelial carcinoma, glioblastoma) [13]. This may be because that telomerase is already epigenetically active in the progenitor cells of tissues that are constantly self-renewing, hence the mutation would not confer any growth advantage [14]. In addition, there is a strong correlation between the presence of the mutations and the age of patients in many types of cancers [4, 15-22].

A two-step mechanism has been proposed for the involvement of TERT-p mutations in tumorigenesis [23]. TERT-p mutations upregulate telomerase activity, but this is not enough to prevent bulk telomere shortening, only delaying senescence. The number of critically short telomeres then increases, causing genomic instability and selective pressure for further upregulation of TERT expression to sustain cell proliferation. Indeed, the telomere length in tumors with TERT-p mutations is shorter than that in normal tissue [24, 25].

TERT-p Mutations in Thyroid Cancer

In 2013, we and others demonstrated that TERT-p mutations were also detected in thyroid cancers, especially in aggressive histological types such as poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) [26, 27]. These mutations were found in all four major histological types derived from thyroid follicular cells, papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), PDTC, and ATC, and were particularly prevalent in the aggressive latter two, but not found in medullary thyroid carcinoma (MTC), which originates from parafollicular C cells. Reported frequencies of TERT-p mutations in thyroid cancer are listed in Table 1. The –124 mutation is more prevalent than the –146 mutation. Shortly after the discovery of the mutations, a number of studies which analyzed the association between the presence of TERT-p mutations and clinicopathological parameters, especially in cases of differentiated thyroid carcinoma (DTC), which consists of PTC and FTC, were done.

Table 1

Frequencies of the TERT promoter mutations in each histological subtype of thyroid cancer

No.	ATC	PDTC	micro PTC	PTC	HCC	FTC	FT-UMP	FTA/benign	MTC	Method	Country	Reference, PMID
1	10/20 (50.0%)	30/58 (52.0%)		18/80 (22.5%)	4/25 (16.0%)					Sanger Sequencing	USA, JAPAN	Landa (2013) JCEM 23833040	[26]
2	23/54 (42.6%)	3/8 (37.5%)		30/257 (11.7%)		9/79 (11.4%)		0/85 (0%)	0/16 (0%)	Sanger Sequencing	USA?	Liu (2013) Endocr Relat Cancer 23766237	[27]
3	2/16 (12.5%)	3/14 (21.4%)		13/169 (7.7%)		9/64 (14.1%)		0/81 (0%)	0/28 (0%)	Sanger Sequencing	Portugal	Vinagre (2013) Nat Commun 23887589	[15]
4	10/20 (50.0%)			13/51 (25.5%)		8/36 (22.2%)			0/37 (0%)	Sanger Sequencing	Sweden	Liu (2014) Oncogene 24141777	[17]
5				46/408 (11.3%)		8/22 (36.4%)		0/44 (0%)		Sanger Sequencing	China	Liu (2014) JCEM 24617711	[18]
6	12/36 (33.3%)	9/31 (29.0%)		25/332 (7.5%)		12/70 (17.1%)				Sanger Sequencing	Portugal	Melo (2014) JCEM 24476079	[16]
7						9/52 (17.3%)		1/58 (1.7%) final diagnosis FTC 3/18 (16.7%) AFTA		Sanger Sequencing	Sweden	Wang (2014) Cancer 24898513	[28]
8				61/507 (12.0%)						Sanger Sequencing	USA	Xing (2014) J Clin Oncol 25024077	[19]
9				36/384 (9.4%)						NGS	USA	TCGA (2014) Cell 25417114	[132]
10									0/42 (0%)	Sanger Sequencing	Sweden	Wang (2014) JCEM 24758186	[133]
11					8/61 (13.1%)					Sanger Sequencing	USA	Chindris (2015) JCEM 25259908	[134]
12				22/182 (12.1%)		8/58 (13.8%)		0/6 (0%)	0/14 (0%)	Sanger Sequencing	Italy?	Muzza (2015) Mol Cell Endo 25448848	[23]
13	41/106 (38.7%)									Sanger Sequencing	USA, China	Shi (2015) JCEM 25584719	[69]
14			19/404 (4.7%)							NGS	Italy	de Biase (2015) Thyroid 26148423	[59]
15				21/121 (17.4%)						Sanger Sequencing	Italy	Gandolfi (2015) Eur J Endocrinol 25583906	[20]
16		6/14 (42.9%)		26/243 (10.7%)	1/3 (33.3%)	1/5 (20%)		0/17 (0%)		Sanger Sequencing	Saudi Arabia	Qasem (2015) Endocr Relat Cancer 26354077	[135]
17	24/33 (72.7%)	34/84 (40.5%)								NGS (MSK-IMPACT)	USA	Landa (2016) JCI 26878173	[70]
18				18/432 (4.2%)		7/119 (5.9%)				Sanger Sequencing	Korea	Song (2016) Cancer 26969876	[31]
19				19/434 (4.4%)						Sanger Sequencing	China	Sun (2016) PLoS One 27064992	[32]
20	7/16 (43.8%)			32/327 (9.8%)		11/66 (16.7%)				Sanger Sequencing	Korea	Kim (2016) Endocr Relat Cancer 27528624	[136]
21				64/1,051 (6.1%)						Sanger Sequencing	USA	Liu (2017) JAMA 27581851	[137]
22				36/357 (10.1%)						Sanger Sequencing	Japan	Matsuse (2017) Sci Rep 28150740	[46]
23	2/6 (33.3%)			21/174 (12.1%)		6/14 (42.9%)				Sanger Sequencing	Portugal, Spain	Melo (2017) JCEM 28323937	[34]
24	98/144 (68.1%)									Sanger Sequencing	France	Bonhomme (2017) Thyroid 28351340	[71]
25	86/118 (72.9%)									Sanger Sequencing	Germany?	Tiedje (2017) Oncotarget 28489587	[72]
26			0/26 (0%)							Sanger Sequencing	Japan	Yabuta (2017) Thyroid 28614984	[68]
27		19/35 (54.3%) fatal PDTC		11/18 (61.1%) fatal PDTC	2/4 (50%) fatal PDTC					NGS	USA	Ibrahimpasic (2017) Clin Cancer Res 28634282	[138]
28				7/355 (2.0%)						NGS	China	Liang (2018) J Pathol 29144541	[139]
29		24/94 (25.5%)								Sanger Sequencing	France	de la Fouchardiere (2018) Eur J Cancer 29413688	[140]
30	127/196? (65%)			285/468? (61%) advanced PTC 0/15? (0%) pediatric PTC	21/35? (59%) advanced HCC	46/65? (71%) advanced FTC				NGS	USA	Pozdeyev (2018) Clin Cancer Res 29615459	[73]
31						19/94 (20.2%)	6/32 (18.8%)	0/42 (0%)		Sanger Sequencing	Sweden	Paulsson (2018) Endcr Relat Cancer 29692346	[35]
32	8/21 (38.1%)	9/21 (42.9%)								Sanger Sequencing	Italy	Romei (2018) Oncol Lett 29805648	[74]
33				8/33 (24.2%) RAIR 0/34 (0%) age and sex matched disease-free PTC						Sanger Sequencing	China	Meng (2019) IUBMB Life 31026111	[53]
34				20/159 (12.6%) by seq plus 3/159 (1.9%) by ddPCR						Sanger Sequencing ddPCR	Japan	Tanaka (2019) Thyroid 31286848	[98]
35				77/568 (13.6%)						Sanger Sequencing	Poland	Trybek (2019) Endocrinology 31305897	[141]
36	12/14 (85.7%)			14/198 (7.1%)	10/40 (25%)	6/34 (17.6%)			0/15 (0%)	Sanger Sequencing	USA	Panebianco (2019) Cancer Medicine 31408918	[6]
37	15/27 (55.6%)	7/15 (46.7%) 11/28 (39.3%) focal ATC/PDTC		15/31 (48.4%) metastatic PTC		9/12 (75%) WI-FTC				NGS	Korea	Yoo (2019) Nat Commun 31235699	[75]
38			4/40 (10.0%) pN1b 0/71 (0%) pN0							NGS	USA	Perera (2019) JCEM 31237614	[60]
39	75/101 (74.3%)									NGS	USA, Australia	Xu (2020) Thyroid 32284020	[76]
40			5/266 (1.9%)	61/712 (8.6%)						Sanger Sequencing	Korea	Song (2020) Cancers 32466217	[61]
41				133/685 (19.4%)						SNaPshot Multiplex system	Japan	Ebina (2020) Cancers 32751594	[47]
42				47/164 (28.7%)						Sanger Sequencing	USA	Liu (2020) J Nucl Med 31375570	[51]
43			0/98 (0%)							Sanger Sequencing	Italy	Sama (2021) Endocrine 32621051	[62]
44				5/133 (3.8%) < 40 mm w/o DM 2/34 (5.9%) > 40 mm w/o DM 10/35 (28.6%) w/ DM		5/16 (31.3%) > 40 mm w/o DM 5/11 (45.5%) w/ DM				Sanger Sequencing	Italy	Vianello (2021) Sci Rep 33790328	[142]
45			16/504 (3.2%)							Sanger Sequencing	Korea	Lee (2021) Surgery 33952391	[63]
46				27/89 (30.3%) > 55 y.o.						ddPCR	Japan	Nakao (2021) Clin Endocrinol 34322882	[79]
47			16/184 (8.7%)							Sanger Sequencing	Saudi Arabia	Parvathareddy (2022) Front Endocrinol 35360077	[64]
48						4/39 (10.3%) MI-FTC 5/24 (20.8%) EA-FTC 5/14 (35.7%) WI-FTC				Sanger Sequencing	Korea	Park (2022) Mod Pathol 34497362	[37]
49			6/1,143 (0.5%)	51/877 (5.8%)						Sanger Sequencing	Korea	Yang (2022) Endocrinol Metab 35864728	[36]
50				20/126 (15.9%) all had RAI therapy						Sanger Sequencing	China	Cao (2022) Eur J Nucl Med Mol Imaging 35501518	[54]
51			39/430 (9.1%)							Sanger Sequencing	Poland	Kuchareczko (2022) Thyroid 35950639	[65]

RAIR; radioiodine refractory, DM; distant metastasis, HCC; Hürthle cell carcinoma, WI-FTC; widely invasive FTC, MI-FTC; minimally invasive FTC, EA-FTC; encapsulated angioinvasive FTC, FT-UMP; follicular tumor of uncertain malignant potential, AFTA; autonomously functioning thyroid adenomas

Clinical and Pathological Implications in DTC

The frequency of TERT-p mutations in DTC was in the range of 10–20% but varied more from report to report (Table 1). As discussed below, TERT-p mutations correlate with tumor aggressiveness and thus may be more frequent in institutions that treat advanced cases.

TERT-p mutations were strongly associated with high-risk clinicopathological characteristics such as age, tumor size, extrathyroidal extension, and distant metastasis, and they are now the best molecular marker so far to assess the risk for recurrence [15-20, 28-39]. Both the number of studies on FTC and the number of analyzed FTC cases are still small compared with those on PTC, hence the evidence for FTC may not be as conclusive as that for PTC. In PTC, the presence of TERT-p mutations is associated with that of the BRAF^V600E mutation, and likewise with the RAS mutation in FTC [27, 38-40]. This means that the probability of having one of these mutations (either TERT-p/BRAF^V600E or TERT-p/RAS) is high in TERT-p mutation-positive tumors. The coexistence of these mutations, especially with the BRAF^V600E mutation, has stronger correlations with the above high-risk clinicopathological parameters [18, 19, 31, 40]. This seems reasonable because many of the ETS family transcription factors are upregulated through the mitogen-activated protein kinase (MAPK) pathway [41], which is strongly activated by the BRAF^V600E mutation.

The BRAF^V600E mutation is the most prevalent driver oncogene found in PTCs and constitutively activates the MAPK signaling pathway, but there is a controversy regarding the clinical significance of this mutation. Many papers especially from Western countries have demonstrated the association between the presence of the BRAF^V600E mutation and tumor aggressiveness and worse prognosis [42, 43]. However, we and others failed to prove such a clinical significance in Japanese PTCs [44-47]. In addition, the prevalence of the BRAF^V600E mutation in Japanese PTCs is higher (70–80%) than that in Western reports (50–60%). There is no definitive explanation for these discrepancies, but a regional difference probably exists [40]. Regarding TERT-p mutations, there seems to be no such difference. The clinicopathological significance of these mutations has been consistently demonstrated everywhere, including Japan [46, 47].

In addition, we have demonstrated that the combination of TERT-p mutations and Ki-67 labeling index, which is another prognostic marker in PTC, enables more precise prediction of prognosis than the TERT-p mutations alone [46]. This was thought to be because TERT-p mutations and Ki-67 represent different biological properties.

Association with Radioactive Iodine Treatment

Radioactive iodine (RAI) treatment is the standard systemic treatment for advanced, persistent, recurrent, and metastatic DTCs, and the response to RAI treatment has a direct impact on prognosis. It has been reported that the presence of the BRAF^V600E mutation is linked to RAI refractoriness (RAI-R) since the activation of the MAPK pathway causes dedifferentiation, leading to impairment of sodium iodide symporter [48-51]. Again, however, there seems to be a regional difference. In a subgroup analysis in the meta-analysis by Luo et al., Asian studies failed to show a significant association between the BRAF^V600E mutation and RAI-R, whereas Western studies demonstrated significance and no heterogeneity [52].

TERT-p mutations have also been demonstrated to be associated with RAI-R by several studies [33, 51-55]. This significance was shown in both Asian and Western studies [52]. The combination of the BRAF^V600E mutation and TERT-p mutations seems to have a stronger link to RAI-R [51, 54, 55]. Interestingly, although the number of studied cases was relatively small, the positive predictive value of TERT-p mutations for RAI-R was very high (most of the cases with TERT-p mutations showed RAI-R), and TERT-p mutations were strongly associated with reduced iodine uptake [33, 53]. On the other hand, however, its sensitivity was not high (there were many RAI-R cases without the TERT-p mutations), suggesting that there are other mechanisms to induce RAI-R. The prevalence of TERT-p mutations in RAI-R cases was 24.2–45.5% [33, 51, 53].

Papillary Thyroid Microcarcinoma

Recently, the category of low-risk papillary thyroid microcarcinoma (PTMC) has gained important clinical significance as active surveillance has been introduced as alternative management for this type of cancer [56-58]. Therefore, the analysis of TERT-p mutations, the best molecular marker to predict prognosis so far, in PTMCs is of great interest. The frequency of TERT-p mutations in PTMCs has been reported to be 0–8.7% [59-66]. These are generally lower than those of all PTCs including advanced cases. One should bear in mind that the detection sensitivity of the next-generation sequencing (NGS), especially deep sequencing used in recent studies, is theoretically higher than that of Sanger sequencing. However, there is no such tendency in the above studies.

At Kuma Hospital in Japan, where a program of active surveillance was started in 1993, and they reported that, over a 10-year observation period for 1,235 cases, 8% had increased in size ≥ 3 mm and 3.8% had novel appearance of lymph node metastasis [67]. We analyzed the specimens from three groups of patients who underwent surgery from the above cohort: 11 patients whose tumors did not grow for >5 years (but who requested surgery), 10 patients whose tumors increased by >4 mm in size, and 5 patients who had novel appearance of lymph node metastasis. TERT-p mutations were not detected by Sanger sequencing in any of the above cases [68]. Although the number of analyzed cases in this study was small, it is important to note that the background population was large and was actually under active surveillance.

The clinical significance of TERT-p mutations in PTMCs is still inconclusive. Four studies reported no association [59, 63, 65, 68]. One study showed an association with age, pT, and pN [36]. A report from Saudi Arabia demonstrated an association with distant metastasis and shorter metastasis-free survival even in multivariate analysis [64]. However, the prevalence of TERT-p mutations in this study was the highest (8.7%), and moreover, distant metastasis and recurrence were found in 6.0% and 15.8% of the cases, respectively, indicating that the PTMC cases in this study may have been unusually aggressive. Overall, the clinical significance of TERT-p mutations in PTMCs remains to be determined.

PDTC and ATC

The frequencies of TERT-p mutations in PDTC and ATC have been reported to be 21.4–51.7% and 12.6–75.0%, respectively, which are much higher than that in DTC [15-17, 26, 27, 39, 69-76]. In some studies, TERT-p mutations were the most prevalent genetic alterations in ATC, suggesting the high importance of these mutations in the most aggressive type of thyroid cancers.

To the best of our knowledge, there are only two studies that analyze the prognostic significance of TERT-p mutations among ATC cases [70, 76]. Overall survival (OS) and OS time have been shown to be shorter in cases with TERT-p mutations, particularly those with concomitant BRAF^V600E or RAS mutations, highlighting the important point that TERT-p mutations make the already highly malignant character of ATCs even more aggressive.

Regarding the anaplastic transformation from DTC, Oishi et al. analyzed the genetic status of 27 ATCs that were accompanied with PTC components [77]. TERT-p mutations were detected in 95% and 91% of the ATC and PTC components, respectively, indicating that its mutational status in both components was virtually identical. These results suggest that TERT-p mutations are associated with a higher risk for anaplastic transformation. Taken together, TERT-p mutations seem to play a key role in accelerating aggressiveness even at different stages.

Clonality and Detection

It has been suggested that TERT-p mutations are a subclonal event in PTCs, whereas they are clonal in PDTCs and ATCs [70, 78]. Average allelic mutant frequency corrected for tumor purity using NGS was 23%, which means that 46% of the PTC cells harbored the mutations. These data prompted us to investigate the detection sensitivity by fine-needle aspiration (FNA), as preoperative detection of TERT-p mutations is likely to be clinically beneficial. We have developed a highly sensitive and specific detection method using droplet digital PCR (ddPCR) [79]. Using this method even for wash solution after FNA, highly concordant results were obtained between FNA and postoperative tissue samples, suggesting that preoperative detection of TERT-p mutations is a useful method for predicting tumor aggressiveness [79] and may enable more precise tailored management. In the same study, the clonality of TERT-p mutations was also assessed in tumors with the BRAF^V600E mutation which was used as a reference of cancer cells. In the majority of the tumors, 80–120% of PTC cells carried the mutation, implying that TERT-p mutations are nearly clonal in most PTCs [79]. We do not yet have a good explanation for the discrepancy from the previous study. However, it should be noted that only relatively older cases (age ≥ 55) were used in our study. The issue of clonality still remains to be explored.

Regulation at the Mutated TERT Promoter

As described in the beginning of this paper, TERT-p mutations create a binding site for the ETS transcription factor family which includes many transcription factors such as ELFs, GABPA, ERG, ETSs, ETVs, and ELKs. First, Bell et al. showed that the most important transcription factor for enhancement of TERT transcription at the TERT-p mutation site was GABPA in glioblastomas [80]. Furthermore, Akincilar et al. found that a long-range interaction between the TERT promoter and 300 kb upstream region through GABPA proteins upregulates TERT transcription [81].

In thyroid cancer cells, Liu et al. demonstrated that BRAF^V600E-MAPK activated FOS, which further upregulated GABPB transcription, and that the GABPA-GABPB complex bound to the mutated TERT promoter and increased TERT expression [82]. Recently, however, several papers have shown that GABPA is not important for TERT promoter-mutated thyroid cancer cells. Because GABPA expression was low in thyroid cancer tissues and also did not correlate well with the activation of the MAPK pathway, several groups performed experiments using thyroid cancer cell lines which suggested that ETV1, ETV4, ETV5 [83], ETV5 [84], ETV1, ELK1, ELF3, and ERF [85] play important roles at the mutated TERT promoter in thyroid cancer. These findings suggest that a definitive mechanism for the enhancement of TERT transcription by TERT-p mutations exists but has yet to be identified. Carefully designed experimental systems will be needed to identify the mechanism, especially in DTCs, where good cell lines are not available.

Other Modes of TERT Activation

As described above, TERT-p mutations are one of the mechanisms by which TERT transcription is reactivated in cancer cells, but there are other modes of re-upregulation of TERT transcription such as genomic rearrangement, promoter methylation, and gene amplification [86–90]. According to the Cancer Genome Atlas (TCGA) and the Pan-Cancer Analysis of Whole Genomes (PCAWG) data, genomic rearrangement, and gene amplification were not detected in PTCs [91, 92]. However, another group identified MTMR12-TERT fusion in PTC (TCGA-BJ-A4O9-01) from the TCGA cohort (TCGA Fusion Gene Database). Subsequently, Yoo et al. identified two other structural rearrangements of TERT in widely invasive FTC [75]. Other studies reported that a small fraction of DTCs and MTCs had gene amplification, and its frequency may be higher in PDTCs and ATCs, but the number of analyzed cases was limited [6, 90, 93, 94]. In thyroid cancer, the most frequent of the other mechanisms of reactivation of TERT transcription is promoter methylation.

In general, DNA methylation at CpG sites in a core promoter region is associated with gene silencing. However, it has been reported that DNA methylation in the TERT promoter region distal to the core promoter causes increased TERT expression. It has been proposed that this is because transcriptional repressors such as CTCF cannot bind to the methylated region [95], leading to transcriptional upregulation.

A connection between TERT-p methylation/transcriptional upregulation and tumor aggressiveness has been demonstrated in childhood brain tumor, prostate cancer, melanoma, and also MTC [88, 89, 94, 96]. This relationship has also been demonstrated in DTC [35, 97]. However, further studies are still needed to determine their difference from TERT-p mutations and the significance of the combination of methylation and mutation.

We investigated the prognostic significance of high TERT expression but without TERT-p mutations in PTCs. Disease-free survival was significantly shorter in cases with high TERT expression compared with expression-free cases [98]. Interestingly, high TERT expression (without TERT-p mutations) was rather common in younger patients and was not associated with the BRAF^V600E mutation or other high-risk clinicopathological parameters. This suggests that high TERT expression may be useful in predicting prognosis in young PTC patients. In addition, the results also suggest that although TERT is highly expressed in both situations (by TERT-p mutations or presumably by promoter methylation), their respective biological significance is different.

PTCs with TERT-p mutations have shorter telomeres than PTCs with the wild-type TERT promoter, even in cases older than 45 years old [17]. Another study demonstrated that TERT re-expression and promoter mutation/methylation/amplification co-occurred in clinically aggressive PTCs [93]. Moreover, significant telomere shortening was observed in these tumors, which was correlated with a higher Ki-67 index. The expression of subtelomeric genes, defined as genes located within a 5 Mb window adjacent to telomeres, was significantly increased in cases with shorter telomeres. Functional annotation revealed that there are many telomere-related genes in the subtelomeric region (e.g., TERT is 1.2 Mb from the 5p terminus). It was suggested that telomere shortening may lead to disruption of the loop structure, resulting in more open chromatin configuration, which may induce mutations in the TERT locus.

Association with SNPs

Several single nucleotide polymorphisms (SNPs) have been reported to affect TERT transcription, albeit controversial [99, 100]. Among them, rs2853669 located at the TERT promoter may have an interaction with TERT-p mutations. TERT-p mutations create a binding site for ETS transcription factors, whereas the rs2853669 disrupts another ETS site on the TERT promoter [101]. It has been reported that rs2853669 suppresses TERT transcriptional activity [3, 100], and that TERT mRNA expression is low in cancer cases carrying the minor allele [101]. Several studies suggest that patients with TERT-p mutations showed poor survival in the absence but not in the presence of rs2853669 in bladder cancer, glioblastoma, and oral squamous cell carcinoma [3, 102-105]. However, there are reports that rs2853669 has no effect on the prognostic impact of TERT-p mutations in glioma including glioblastoma [101, 106]. Conversely, it has been also reported that the combination of rs2853669 and the TERT-p mutation increased TERT expression and worsened prognosis in glioblastoma and liver cancer [107, 108]. There are inconsistencies even within the same type of cancer. In thyroid cancer, the coexistence of rs2853669 and the TERT-p mutation has been reported to be associated with an increase in tumor size [109, 110]. Overall, however, the biological and clinical significance of rs2853669, especially in relation to TERT-p mutations, is still inconclusive, and further studies are definitely needed.

Possible Mechanisms to Enhance Tumor Aggressiveness

It is easy to understand how telomerase activity would be important for overcoming the Hayflick limit and acquiring the infinite proliferative capacity of cancer cells. While it could well be associated with a faster growth rate, telomere elongation cannot adequately explain the other higher malignant properties such as invasion and metastasis. Indeed, novel TERT functions other than the above canonical telomerase activity have long been proposed [111].

The earliest evidence for a telomere-independent role for Tert comes from studies in mice. Since mice have very long telomeres (20–50 kb) compared to humans (5–10 kb), increased telomerase activity is not required in mouse tumors to prevent telomere shortening and replicative senescence. However, Tert was found to be upregulated in mouse breast cancer [112] and skin cancer [113], suggesting that Tert may also play a role other than telomere maintenance. Overexpression of Tert in mouse thymocytes caused T-cell lymphoma without affecting telomere length, also supporting a telomerase-independent role for Tert [114]. In addition, in the same study, it was shown that chromosomal instability was enhanced in cells with Tert overexpression after gamma irradiation, suggesting that Tert interferes with DNA damage response [114].

In normal cells, Sarin et al. showed that conditional expression of Tert in mouse skin epithelium induced proliferation of hair follicle stem cells, and that it was independent of telomerase activity [115]. The same group also demonstrated that a mutant Tert lacking RT function enhanced keratinocyte proliferation and activated hair follicle stem cells through Wnt and Myc [116]. In human cells, Smith et al. demonstrated that ectopic expression of TERT in human mammary epithelial cells enhanced cell proliferation through EGFR [117]. Hrdlickova et al. found that the overexpression of a naturally occurring alternative splicing variant (Δ4–13) of TERT accelerated the growth of several types of human cells without telomerase activity by stimulating the WNT signaling [118]. Telomerase-inactive mutant TERT has also been reported to be implicated in anti-apoptosis [119, 120].

TERT has also been reported to regulate angiogenesis. Zhou et al. showed that TERT activated VEGF transcription in WI-38 and HeLa cells, which was independent of telomerase activity [121]. Conversely, other papers demonstrated that VEGF upregulated TERT expression [122, 123], suggesting the existence of a possible positive feedback loop.

TERT has also been shown to interact with other molecules that are important in tumor progression. Park et al. reported that TERT interacted with BRG1, a chromatin-remodelling factor, and upregulated Wnt/β-catenin target genes [124]. Ghosh et al. demonstrated that TERT associated with RELA (P65) and induced NF-κB-dependent gene expression such as IL-6, IL-8, and TNF-α [125]. These cytokines play pivotal roles in inflammation, anti-apoptosis, and cancer progression. Interestingly, both Wnt/β-catenin and NF-κB have been shown to upregulate TERT transcription [126, 127], suggesting that this feed-forward loop may maintain the critical activation levels of these pathways.

An interesting novel enzymatic activity of TERT has also been reported. Phosphorylated TERT at threonine 249 by CDK1 acquired RNA dependent RNA polymerase (RdRP) activity. The complementary RNA of the mRNA of FOXO4, a tumor suppressor gene, was synthesized by TERT-RdRP, and this dsRNA complex was degraded, resulting in the downregulation of FOXO4 expression [128]. As FOXO4 has a tumor suppressor role, reduced FOXO4 expression is associated with tumor progression.

TERT has been shown to be involved in DNA damage response. Masutomi et al. reported that damage response to DNA double-strand breaks was impaired in cells lacking TERT. This is not recovered by a catalytically inactive TERT mutant, suggesting that telomerase activity may be required for this response [129]. Liu et al. used an irreversible TERT inhibitor, NU-1, and demonstrated that inhibiting TERT sensitized cancer cells to chemotherapy and ionizing radiation [130]. NU-1 delayed DNA repair of double-strand breaks, promoting cell senescence. This NU-1 effect was not observed in cells without TERT expression.

In thyroid cancer, the association between TERT and autophagy was demonstrated. Overexpression of TERT activated autophagy, which was suggested to be one of the mechanisms of tumor aggressiveness [131].

These findings indicate that TERT has non-canonical functions that play important roles in cancer progression. As TERT-p mutations induce TERT expression, these mechanisms are likely to be involved in enhancing tumor aggressiveness in thyroid cancer.

Conclusions

TERT-p mutations are strongly associated with tumor aggressiveness, response to treatment, and prognosis in thyroid cancer, at least in DTC. They are thus expected to be a good molecular marker for tailored management of the condition and could also be a novel therapeutic target for aggressive thyroid cancers.

Disclosure

The authors declare no conflict of interests.

References

• 1   Huang  FW,  Hodis  E,  Xu  MJ,  Kryukov  GV,  Chin  L, et al. (2013) Highly recurrent TERT promoter mutations in human melanoma. Science 339: 957–959.
• 2   Horn  S,  Figl  A,  Rachakonda  PS,  Fischer  C,  Sucker  A, et al. (2013) TERT promoter mutations in familial and sporadic melanoma. Science 339: 959–961.
• 3   Rachakonda  PS,  Hosen  I,  de Verdier  PJ,  Fallah  M,  Heidenreich  B, et al. (2013) TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism. Proc Natl Acad Sci U S A 110: 17426–17431.
• 4   Chen  C,  Han  S,  Meng  L,  Li  Z,  Zhang  X, et al. (2014) TERT promoter mutations lead to high transcriptional activity under hypoxia and temozolomide treatment and predict poor prognosis in gliomas. PLoS One 9: e100297.
• 5   Andres-Lencina  JJ,  Rachakonda  S,  Garcia-Casado  Z,  Srinivas  N,  Skorokhod  A, et al. (2019) TERT promoter mutation subtypes and survival in stage I and II melanoma patients. Int J Cancer 144: 1027–1036.
• 6   Panebianco  F,  Nikitski  AV,  Nikiforova  MN,  Nikiforov  YE (2019) Spectrum of TERT promoter mutations and mechanisms of activation in thyroid cancer. Cancer Med 8: 5831–5839.
• 7   Hirokawa  T,  Arimasu  Y,  Chiba  T,  Nakazato  Y,  Fujiwara  M, et al. (2020) Regulatory single nucleotide polymorphism increases TERT promoter activity in thyroid carcinoma cells. Pathobiology 87: 338–344.
• 8   Huang  DS,  Wang  Z,  He  XJ,  Diplas  BH,  Yang  R, et al. (2015) Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation. Eur J Cancer 51: 969–976.
• 9   Xu  T,  Cheng  D,  Zhao  Y,  Zhang  J,  Zhu  X, et al. (2021) Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene. Proc Natl Acad Sci U S A 118: e2019043118.
• 10   Heidenreich  B,  Rachakonda  PS,  Hosen  I,  Volz  F,  Hemminki  K, et al. (2015) TERT promoter mutations and telomere length in adult malignant gliomas and recurrences. Oncotarget 6: 10617–10633.
• 11   Cancer Genome Atlas  Network (2015) Genomic classification of cutaneous melanoma. Cell 161: 1681–1696.
• 12   Li  Y,  Zhou  QL,  Sun  W,  Chandrasekharan  P,  Cheng  HS, et al. (2015) Non-canonical NF-kappaB signalling and ETS1/2 cooperatively drive C250T mutant TERT promoter activation. Nat Cell Biol 17: 1327–1338.
• 13   Killela  PJ,  Reitman  ZJ,  Jiao  Y,  Bettegowda  C,  Agrawal  N, et al. (2013) 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 110: 6021–6026.
• 14   Lorbeer  FK,  Hockemeyer  D (2020) TERT promoter mutations and telomeres during tumorigenesis. Curr Opin Genet Dev 60: 56–62.
• 15   Vinagre  J,  Almeida  A,  Populo  H,  Batista  R,  Lyra  J, et al. (2013) Frequency of TERT promoter mutations in human cancers. Nat Commun 4: 2185.
• 16   Melo  M,  da Rocha  AG,  Vinagre  J,  Batista  R,  Peixoto  J, et al. (2014) TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas. J Clin Endocrinol Metab 99: E754–E765.
• 17   Liu  T,  Wang  N,  Cao  J,  Sofiadis  A,  Dinets  A, et al. (2014) The age- and shorter telomere-dependent TERT promoter mutation in follicular thyroid cell-derived carcinomas. Oncogene 33: 4978–4984.
• 18   Liu  X,  Qu  S,  Liu  R,  Sheng  C,  Shi  X, et al. (2014) TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer. J Clin Endocrinol Metab 99: E1130–E1136.
• 19   Xing  M,  Liu  R,  Liu  X,  Murugan  AK,  Zhu  G, et al. (2014) BRAF V600E and TERT promoter mutations cooperatively identify the most aggressive papillary thyroid cancer with highest recurrence. J Clin Oncol 32: 2718–2726.
• 20   Gandolfi  G,  Ragazzi  M,  Frasoldati  A,  Piana  S,  Ciarrocchi  A, et al. (2015) TERT promoter mutations are associated with distant metastases in papillary thyroid carcinoma. Eur J Endocrinol 172: 403–413.
• 21   Chen  YL,  Jeng  YM,  Chang  CN,  Lee  HJ,  Hsu  HC, et al. (2014) TERT promoter mutation in resectable hepatocellular carcinomas: a strong association with hepatitis C infection and absence of hepatitis B infection. Int J Surg 12: 659–665.
• 22   Huang  HN,  Chiang  YC,  Cheng  WF,  Chen  CA,  Lin  MC, et al. (2015) Molecular alterations in endometrial and ovarian clear cell carcinomas: clinical impacts of telomerase reverse transcriptase promoter mutation. Mod Pathol 28: 303–311.
• 23   Chiba  K,  Lorbeer  FK,  Shain  AH,  McSwiggen  DT,  Schruf  E, et al. (2017) Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism. Science 357: 1416–1420.
• 24   Ceccarelli  M,  Barthel  FP,  Malta  TM,  Sabedot  TS,  Salama  SR, et al. (2016) Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell 164: 550–563.
• 25   Hayward  NK,  Wilmott  JS,  Waddell  N,  Johansson  PA,  Field  MA, et al. (2017) Whole-genome landscapes of major melanoma subtypes. Nature 545: 175–180.
• 26   Landa  I,  Ganly  I,  Chan  TA,  Mitsutake  N,  Matsuse  M, et al. (2013) Frequent somatic TERT promoter mutations in thyroid cancer: higher prevalence in advanced forms of the disease. J Clin Endocrinol Metab 98: E1562–E1566.
• 27   Liu  X,  Bishop  J,  Shan  Y,  Pai  S,  Liu  D, et al. (2013) Highly prevalent TERT promoter mutations in aggressive thyroid cancers. Endocr Relat Cancer 20: 603–610.
• 28   Wang  N,  Liu  T,  Sofiadis  A,  Juhlin  CC,  Zedenius  J, et al. (2014) TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA. Cancer 120: 2965–2979.
• 29   Liu  R,  Xing  M (2014) Diagnostic and prognostic TERT promoter mutations in thyroid fine-needle aspiration biopsy. Endocr Relat Cancer 21: 825–830.
• 30   Muzza  M,  Colombo  C,  Rossi  S,  Tosi  D,  Cirello  V, et al. (2015) Telomerase in differentiated thyroid cancer: promoter mutations, expression and localization. Mol Cell Endocrinol 399: 288–295.
• 31   Song  YS,  Lim  JA,  Choi  H,  Won  JK,  Moon  JH, et al. (2016) Prognostic effects of TERT promoter mutations are enhanced by coexistence with BRAF or RAS mutations and strengthen the risk prediction by the ATA or TNM staging system in differentiated thyroid cancer patients. Cancer 122: 1370–1379.
• 32   Sun  J,  Zhang  J,  Lu  J,  Gao  J,  Ren  X, et al. (2016) BRAF V600E and TERT promoter mutations in papillary thyroid carcinoma in Chinese patients. PLoS One 11: e0153319.
• 33   Yang  X,  Li  J,  Li  X,  Liang  Z,  Gao  W, et al. (2017) TERT promoter mutation predicts radioiodine-refractory character in distant metastatic differentiated thyroid cancer. J Nucl Med 58: 258–265.
• 34   Melo  M,  Gaspar da Rocha  A,  Batista  R,  Vinagre  J,  Martins  MJ, et al. (2017) TERT, BRAF, and NRAS in primary thyroid cancer and metastatic disease. J Clin Endocrinol Metab 102: 1898–1907.
• 35   Paulsson  JO,  Mu  N,  Shabo  I,  Wang  N,  Zedenius  J, et al. (2018) TERT aberrancies: a screening tool for malignancy in follicular thyroid tumours. Endocr Relat Cancer 25: 723–733.
• 36   Yang  H,  Park  H,  Ryu  HJ,  Heo  J,  Kim  JS, et al. (2022) Frequency of TERT promoter mutations in real-world analysis of 2,092 thyroid carcinoma patients. Endocrinol Metab (Seoul) 37: 652–663.
• 37   Park  H,  Shin  HC,  Yang  H,  Heo  J,  Ki  CS, et al. (2022) Molecular classification of follicular thyroid carcinoma based on TERT promoter mutations. Mod Pathol 35: 186–192.
• 38   Liu  R,  Xing  M (2016) TERT promoter mutations in thyroid cancer. Endocr Relat Cancer 23: R143–R155.
• 39   Alzahrani  AS,  Alsaadi  R,  Murugan  AK,  Sadiq  BB (2016) TERT promoter mutations in thyroid cancer. Horm Cancer 7: 165–177.
• 40   Chen  B,  Shi  Y,  Xu  Y,  Zhang  J (2021) The predictive value of coexisting BRAFV600E and TERT promoter mutations on poor outcomes and high tumour aggressiveness in papillary thyroid carcinoma: a systematic review and meta-analysis. Clin Endocrinol (Oxf) 94: 731–742.
• 41   Selvaraj  N,  Kedage  V,  Hollenhorst  PC (2015) Comparison of MAPK specificity across the ETS transcription factor family identifies a high-affinity ERK interaction required for ERG function in prostate cells. Cell Commun Signal 13: 12.
• 42   Xing  M (2007) BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 28: 742–762.
• 43   Tufano  RP,  Teixeira  GV,  Bishop  J,  Carson  KA,  Xing  M (2012) BRAF mutation in papillary thyroid cancer and its value in tailoring initial treatment: a systematic review and meta-analysis. Medicine (Baltimore) 91: 274–286.
• 44   Ito  Y,  Yoshida  H,  Maruo  R,  Morita  S,  Takano  T, et al. (2009) BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients. Endocr J 56: 89–97.
• 45   Mitsutake  N,  Fukushima  T,  Matsuse  M,  Rogounovitch  T,  Saenko  V, et al. (2015) BRAF(V600E) mutation is highly prevalent in thyroid carcinomas in the young population in Fukushima: a different oncogenic profile from Chernobyl. Sci Rep 5: 16976.
• 46   Matsuse  M,  Yabuta  T,  Saenko  V,  Hirokawa  M,  Nishihara  E, et al. (2017) TERT promoter mutations and Ki-67 labeling index as a prognostic marker of papillary thyroid carcinomas: combination of two independent factors. Sci Rep 7: 41752.
• 47   Ebina  A,  Togashi  Y,  Baba  S,  Sato  Y,  Sakata  S, et al. (2020) TERT promoter mutation and extent of thyroidectomy in patients with 1–4 cm intrathyroidal papillary carcinoma. Cancers (Basel) 12: 2115.
• 48   Mitsutake  N,  Knauf  JA,  Mitsutake  S,  Mesa  C Jr,  Zhang  L, et al. (2005) Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells. Cancer Res 65: 2465–2473.
• 49   Liu  D,  Hu  S,  Hou  P,  Jiang  D,  Condouris  S, et al. (2007) Suppression of BRAF/MEK/MAP kinase pathway restores expression of iodide-metabolizing genes in thyroid cells expressing the V600E BRAF mutant. Clin Cancer Res 13: 1341–1349.
• 50   Chakravarty  D,  Santos  E,  Ryder  M,  Knauf  JA,  Liao  XH, et al. (2011) Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation. J Clin Invest 121: 4700–4711.
• 51   Liu  J,  Liu  R,  Shen  X,  Zhu  G,  Li  B, et al. (2020) The genetic duet of BRAF V600E and TERT promoter mutations robustly predicts loss of radioiodine avidity in recurrent papillary thyroid cancer. J Nucl Med 61: 177–182.
• 52   Luo  Y,  Jiang  H,  Xu  W,  Wang  X,  Ma  B, et al. (2020) Clinical, pathological, and molecular characteristics correlating to the occurrence of radioiodine refractory differentiated thyroid carcinoma: a systematic review and meta-analysis. Front Oncol 10: 549882.
• 53   Meng  Z,  Matsuse  M,  Saenko  V,  Yamashita  S,  Ren  P, et al. (2019) TERT promoter mutation in primary papillary thyroid carcinoma lesions predicts absent or lower (131) i uptake in metastases. IUBMB Life 71: 1030–1040.
• 54   Cao  J,  Zhu  X,  Sun  Y,  Li  X,  Yun  C, et al. (2022) The genetic duet of BRAF V600E and TERT promoter mutations predicts the poor curative effect of radioiodine therapy in papillary thyroid cancer. Eur J Nucl Med Mol Imaging 49: 3470–3481.
• 55   Soe  MH,  Chiang  JM,  Flavell  RR,  Khanafshar  E,  Mendoza  L, et al. (2022) Non-iodine-avid disease is highly prevalent in distant metastatic differentiated thyroid cancer with papillary histology. J Clin Endocrinol Metab 107: e3206–e3216.
• 56   Ito  Y,  Uruno  T,  Nakano  K,  Takamura  Y,  Miya  A, et al. (2003) An observation trial without surgical treatment in patients with papillary microcarcinoma of the thyroid. Thyroid 13: 381–387.
• 57   Sugitani  I,  Toda  K,  Yamada  K,  Yamamoto  N,  Ikenaga  M, et al. (2010) Three distinctly different kinds of papillary thyroid microcarcinoma should be recognized: our treatment strategies and outcomes. World J Surg 34: 1222–1231.
• 58   Horiguchi  K,  Yoshida  Y,  Iwaku  K,  Emoto  N,  Kasahara  T, et al. (2021) Position paper from the Japan Thyroid Association task force on the management of low-risk papillary thyroid microcarcinoma (T1aN0M0) in adults. Endocr J 68: 763–780.
• 59   de Biase  D,  Gandolfi  G,  Ragazzi  M,  Eszlinger  M,  Sancisi  V, et al. (2015) TERT promoter mutations in papillary thyroid microcarcinomas. Thyroid 25: 1013–1019.
• 60   Perera  D,  Ghossein  R,  Camacho  N,  Senbabaoglu  Y,  Seshan  V, et al. (2019) Genomic and transcriptomic characterization of papillary microcarcinomas with lateral neck lymph node metastases. J Clin Endocrinol Metab 104: 4889–4899.
• 61   Song  YS,  Kang  BH,  Lee  S,  Yoo  SK,  Choi  YS, et al. (2020) Genomic and transcriptomic characteristics according to size of papillary thyroid microcarcinoma. Cancers (Basel) 12: 1345.
• 62   Sama  MT,  Grosso  E,  Mele  C,  Laurora  S,  Monzeglio  O, et al. (2021) Molecular characterisation and clinical correlation of papillary thyroid microcarcinoma. Endocrine 71: 149–157.
• 63   Lee  J,  Ha  EJ,  Roh  J,  Kim  HK (2021) Presence of TERT +/– BRAF V600E mutation is not a risk factor for the clinical management of patients with papillary thyroid microcarcinoma. Surgery 170: 743–747.
• 64   Parvathareddy  SK,  Siraj  AK,  Iqbal  K,  Qadri  Z,  Ahmed  SO, et al. (2022) TERT promoter mutations are an independent predictor of distant metastasis in middle eastern papillary thyroid microcarcinoma. Front Endocrinol (Lausanne) 13: 808298.
• 65   Kuchareczko  A,  Kopczynski  J,  Kowalik  A,  Hincza-Nowak  K,  Walczyk  A, et al. (2022) A significance of concomitant BRAF(V600E) and TERT mutations in polish patients with papillary thyroid microcarcinoma: a retrospective cohort study based on 430 cases. Thyroid 32: 1372–1381.
• 66   Yu  FX,  Hu  MX,  Zhao  HX,  Niu  LJ,  Rong  XY, et al. (2019) Precise detection of gene mutations in fine-needle aspiration specimens of the papillary thyroid microcarcinoma using next-generation sequencing. Int J Endocrinol 2019: 4723958.
• 67   Ito  Y,  Miyauchi  A,  Kihara  M,  Higashiyama  T,  Kobayashi  K, et al. (2014) Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation. Thyroid 24: 27–34.
• 68   Yabuta  T,  Matsuse  M,  Hirokawa  M,  Yamashita  S,  Mitsutake  N, et al. (2017) TERT promoter mutations were not found in papillary thyroid microcarcinomas that showed disease progression on active surveillance. Thyroid 27: 1206–1207.
• 69   Shi  X,  Liu  R,  Qu  S,  Zhu  G,  Bishop  J, et al. (2015) Association of TERT promoter mutation 1,295,228 C > T with BRAF V600E mutation, older patient age, and distant metastasis in anaplastic thyroid cancer. J Clin Endocrinol Metab 100: E632–E637.
• 70   Landa  I,  Ibrahimpasic  T,  Boucai  L,  Sinha  R,  Knauf  JA, et al. (2016) Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. J Clin Invest 126: 1052–1066.
• 71   Bonhomme  B,  Godbert  Y,  Perot  G,  Al Ghuzlan  A,  Bardet  S, et al. (2017) Molecular pathology of anaplastic thyroid carcinomas: a retrospective study of 144 cases. Thyroid 27: 682–692.
• 72   Tiedje  V,  Ting  S,  Herold  T,  Synoracki  S,  Latteyer  S, et al. (2017) NGS based identification of mutational hotspots for targeted therapy in anaplastic thyroid carcinoma. Oncotarget 8: 42613–42620.
• 73   Pozdeyev  N,  Gay  LM,  Sokol  ES,  Hartmaier  R,  Deaver  KE, et al. (2018) Genetic analysis of 779 advanced differentiated and anaplastic thyroid cancers. Clin Cancer Res 24: 3059–3068.
• 74   Romei  C,  Tacito  A,  Molinaro  E,  Piaggi  P,  Cappagli  V, et al. (2018) Clinical, pathological and genetic features of anaplastic and poorly differentiated thyroid cancer: a single institute experience. Oncol Lett 15: 9174–9182.
• 75   Yoo  SK,  Song  YS,  Lee  EK,  Hwang  J,  Kim  HH, et al. (2019) Integrative analysis of genomic and transcriptomic characteristics associated with progression of aggressive thyroid cancer. Nat Commun 10: 2764.
• 76   Xu  B,  Fuchs  T,  Dogan  S,  Landa  I,  Katabi  N, et al. (2020) Dissecting anaplastic thyroid carcinoma: a comprehensive clinical, histologic, immunophenotypic, and molecular study of 360 cases. Thyroid 30: 1505–1517.
• 77   Oishi  N,  Kondo  T,  Ebina  A,  Sato  Y,  Akaishi  J, et al. (2017) Molecular alterations of coexisting thyroid papillary carcinoma and anaplastic carcinoma: identification of TERT mutation as an independent risk factor for transformation. Mod Pathol 30: 1527–1537.
• 78   Xu  B,  Ghossein  R (2016) Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma. Endocr Pathol 27: 205–212.
• 79   Nakao  T,  Matsuse  M,  Saenko  V,  Rogounovitch  T,  Tanaka  A, et al. (2021) Preoperative detection of the TERT promoter mutations in papillary thyroid carcinomas. Clin Endocrinol (Oxf) 95: 790–799.
• 80   Bell  RJ,  Rube  HT,  Kreig  A,  Mancini  A,  Fouse  SD, et al. (2015) Cancer. The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer. Science 348: 1036–1039.
• 81   Akincilar  SC,  Khattar  E,  Boon  PL,  Unal  B,  Fullwood  MJ, et al. (2016) Long-range chromatin interactions drive mutant TERT promoter activation. Cancer Discov 6: 1276–1291.
• 82   Liu  R,  Zhang  T,  Zhu  G,  Xing  M (2018) Regulation of mutant TERT by BRAF V600E/MAP kinase pathway through FOS/GABP in human cancer. Nat Commun 9: 579.
• 83   Song  YS,  Yoo  SK,  Kim  HH,  Jung  G,  Oh  AR, et al. (2019) Interaction of BRAF-induced ETS factors with mutant TERT promoter in papillary thyroid cancer. Endocr Relat Cancer 26: 629–641.
• 84   Bullock  M,  Lim  G,  Zhu  Y,  Aberg  H,  Kurdyukov  S, et al. (2019) ETS factor ETV5 activates the mutant telomerase reverse transcriptase promoter in thyroid cancer. Thyroid 29: 1623–1633.
• 85   Thornton  CEM,  Hao  J,  Tamarapu  PP,  Landa  I (2022) Multiple ETS factors participate in the transcriptional control of TERT mutant promoter in thyroid cancers. Cancers (Basel) 14: 357.
• 86   Davis  CF,  Ricketts  CJ,  Wang  M,  Yang  L,  Cherniack  AD, et al. (2014) The somatic genomic landscape of chromophobe renal cell carcinoma. Cancer Cell 26: 319–330.
• 87   Peifer  M,  Hertwig  F,  Roels  F,  Dreidax  D,  Gartlgruber  M, et al. (2015) Telomerase activation by genomic rearrangements in high-risk neuroblastoma. Nature 526: 700–704.
• 88   Castelo-Branco  P,  Choufani  S,  Mack  S,  Gallagher  D,  Zhang  C, et al. (2013) Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. Lancet Oncol 14: 534–542.
• 89   Castelo-Branco  P,  Leao  R,  Lipman  T,  Campbell  B,  Lee  D, et al. (2016) A cancer specific hypermethylation signature of the TERT promoter predicts biochemical relapse in prostate cancer: a retrospective cohort study. Oncotarget 7: 57726–57736.
• 90   Gupta  S,  Vanderbilt  CM,  Lin  YT,  Benhamida  JK,  Jungbluth  AA, et al. (2021) A pan-cancer study of somatic TERT promoter mutations and amplification in 30,773 tumors profiled by clinical genomic sequencing. J Mol Diagn 23: 253–263.
• 91   Barthel  FP,  Wei  W,  Tang  M,  Martinez-Ledesma  E,  Hu  X, et al. (2017) Systematic analysis of telomere length and somatic alterations in 31 cancer types. Nat Genet 49: 349–357.
• 92   Sieverling  L,  Hong  C,  Koser  SD,  Ginsbach  P,  Kleinheinz  K, et al. (2020) Genomic footprints of activated telomere maintenance mechanisms in cancer. Nat Commun 11: 733.
• 93   Montero-Conde  C,  Leandro-Garcia  LJ,  Martinez-Montes  AM,  Martinez  P,  Moya  FJ, et al. (2022) Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers. Clin Transl Med 12: e1001.
• 94   Wang  N,  Kjellin  H,  Sofiadis  A,  Fotouhi  O,  Juhlin  CC, et al. (2016) Genetic and epigenetic background and protein expression profiles in relation to telomerase activation in medullary thyroid carcinoma. Oncotarget 7: 21332–21346.
• 95   Renaud  S,  Loukinov  D,  Abdullaev  Z,  Guilleret  I,  Bosman  FT, et al. (2007) Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene. Nucleic Acids Res 35: 1245–1256.
• 96   Fan  Y,  Lee  S,  Wu  G,  Easton  J,  Yergeau  D, et al. (2016) Telomerase expression by aberrant methylation of the TERT promoter in melanoma arising in giant congenital nevi. J Invest Dermatol 136: 339–342.
• 97   Li  JJ,  Zheng  PCJ,  Wang  YZ (2017) The correlations between DNA methylation and polymorphisms in the promoter region of the human telomerase reverse transcriptase (hTERT) gene with postoperative recurrence in patients with thyroid carcinoma (TC). World J Surg Oncol 15: 114.
• 98   Tanaka  A,  Matsuse  M,  Saenko  V,  Nakao  T,  Yamanouchi  K, et al. (2019) TERT mRNA expression as a novel prognostic marker in papillary thyroid carcinomas. Thyroid 29: 1105–1114.
• 99   Bojesen  SE,  Pooley  KA,  Johnatty  SE,  Beesley  J,  Michailidou  K, et al. (2013) Multiple independent variants at the TERT locus are associated with telomere length and risks of breast and ovarian cancer. Nat Genet 45: 371–384.
• 100   Helbig  S,  Wockner  L,  Bouendeu  A,  Hille-Betz  U,  McCue  K, et al. (2017) Functional dissection of breast cancer risk-associated TERT promoter variants. Oncotarget 8: 67203–67217.
• 101   Labussiere  M,  Di Stefano  AL,  Gleize  V,  Boisselier  B,  Giry  M, et al. (2014) TERT promoter mutations in gliomas, genetic associations and clinico-pathological correlations. Br J Cancer 111: 2024–2032.
• 102   Simon  M,  Hosen  I,  Gousias  K,  Rachakonda  S,  Heidenreich  B, et al. (2015) TERT promoter mutations: a novel independent prognostic factor in primary glioblastomas. Neuro Oncol 17: 45–52.
• 103   Spiegl-Kreinecker  S,  Lotsch  D,  Ghanim  B,  Pirker  C,  Mohr  T, et al. (2015) Prognostic quality of activating TERT promoter mutations in glioblastoma: interaction with the rs2853669 polymorphism and patient age at diagnosis. Neuro Oncol 17: 1231–1240.
• 104   Giunco  S,  Boscolo-Rizzo  P,  Rampazzo  E,  Tirelli  G,  Alessandrini  L, et al. (2021) TERT promoter mutations and rs2853669 polymorphism: useful markers for clinical outcome stratification of patients with oral cavity squamous cell carcinoma. Front Oncol 11: 782658.
• 105   Batista  R,  Cruvinel-Carloni  A,  Vinagre  J,  Peixoto  J,  Catarino  TA, et al. (2016) The prognostic impact of TERT promoter mutations in glioblastomas is modified by the rs2853669 single nucleotide polymorphism. Int J Cancer 139: 414–423.
• 106   Nencha  U,  Rahimian  A,  Giry  M,  Sechi  A,  Mokhtari  K, et al. (2016) TERT promoter mutations and rs2853669 polymorphism: prognostic impact and interactions with common alterations in glioblastomas. J Neurooncol 126: 441–446.
• 107   Ko  E,  Seo  HW,  Jung  ES,  Kim  BH,  Jung  G (2016) The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer. Oncotarget 7: 684–699.
• 108   Mosrati  MA,  Malmstrom  A,  Lysiak  M,  Krysztofiak  A,  Hallbeck  M, et al. (2015) TERT promoter mutations and polymorphisms as prognostic factors in primary glioblastoma. Oncotarget 6: 16663–16673.
• 109   Hirokawa  T,  Arimasu  Y,  Nakazato  Y,  Chiba  T,  Fujiwara  M, et al. (2020) Effect of single-nucleotide polymorphism in TERT promoter on follicular thyroid tumor development. Pathol Int 70: 210–216.
• 110   Hirokawa  T,  Arimasu  Y,  Chiba  T,  Fujiwara  M,  Kamma  H (2020) Clinicopathological significance of the single nucleotide polymorphism, rs2853669 within the TERT promoter in papillary thyroid carcinoma. Pathol Int 70: 217–223.
• 111   Low  KC,  Tergaonkar  V (2013) Telomerase: central regulator of all of the hallmarks of cancer. Trends Biochem Sci 38: 426–434.
• 112   Broccoli  D,  Godley  LA,  Donehower  LA,  Varmus  HE,  de Lange  T (1996) Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation. Mol Cell Biol 16: 3765–3772.
• 113   Bednarek  A,  Budunova  I,  Slaga  TJ,  Aldaz  CM (1995) Increased telomerase activity in mouse skin premalignant progression. Cancer Res 55: 4566–4569.
• 114   Canela  A,  Martin-Caballero  J,  Flores  JM,  Blasco  MA (2004) Constitutive expression of tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice. Mol Cell Biol 24: 4275–4293.
• 115   Sarin  KY,  Cheung  P,  Gilison  D,  Lee  E,  Tennen  RI, et al. (2005) Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature 436: 1048–1052.
• 116   Choi  J,  Southworth  LK,  Sarin  KY,  Venteicher  AS,  Ma  W, et al. (2008) TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program. PLoS Genet 4: e10.
• 117   Smith  LL,  Coller  HA,  Roberts  JM (2003) Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat Cell Biol 5: 474–479.
• 118   Hrdlickova  R,  Nehyba  J,  Bose  HR Jr (2012) Alternatively spliced telomerase reverse transcriptase variants lacking telomerase activity stimulate cell proliferation. Mol Cell Biol 32: 4283–4296.
• 119   Rahman  R,  Latonen  L,  Wiman  KG (2005) hTERT antagonizes p53-induced apoptosis independently of telomerase activity. Oncogene 24: 1320–1327.
• 120   Del Bufalo  D,  Rizzo  A,  Trisciuoglio  D,  Cardinali  G,  Torrisi  MR, et al. (2005) Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function. Cell Death Differ 12: 1429–1438.
• 121   Zhou  L,  Zheng  D,  Wang  M,  Cong  YS (2009) Telomerase reverse transcriptase activates the expression of vascular endothelial growth factor independent of telomerase activity. Biochem Biophys Res Commun 386: 739–743.
• 122   Zaccagnini  G,  Gaetano  C,  Della Pietra  L,  Nanni  S,  Grasselli  A, et al. (2005) Telomerase mediates vascular endothelial growth factor-dependent responsiveness in a rat model of hind limb ischemia. J Biol Chem 280: 14790–14798.
• 123   Bermudez  Y,  Yang  H,  Saunders  BO,  Cheng  JQ,  Nicosia  SV, et al. (2007) VEGF- and LPA-induced telomerase in human ovarian cancer cells is Sp1-dependent. Gynecol Oncol 106: 526–537.
• 124   Park  JI,  Venteicher  AS,  Hong  JY,  Choi  J,  Jun  S, et al. (2009) Telomerase modulates Wnt signalling by association with target gene chromatin. Nature 460: 66–72.
• 125   Ghosh  A,  Saginc  G,  Leow  SC,  Khattar  E,  Shin  EM, et al. (2012) Telomerase directly regulates NF-κB-dependent transcription. Nat Cell Biol 14: 1270–1281.
• 126   Yin  L,  Hubbard  AK,  Giardina  C (2000) NF-kappa B regulates transcription of the mouse telomerase catalytic subunit. J Biol Chem 275: 36671–36675.
• 127   Zhang  Y,  Toh  L,  Lau  P,  Wang  X (2012) Human telomerase reverse transcriptase (hTERT) is a novel target of the Wnt/β-catenin pathway in human cancer. J Biol Chem 287: 32494–32511.
• 128   Yasukawa  M,  Ando  Y,  Yamashita  T,  Matsuda  Y,  Shoji  S, et al. (2020) CDK1 dependent phosphorylation of hTERT contributes to cancer progression. Nat Commun 11: 1557.
• 129   Masutomi  K,  Possemato  R,  Wong  JM,  Currier  JL,  Tothova  Z, et al. (2005) The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc Natl Acad Sci U S A 102: 8222–8227.
• 130   Liu  Y,  Betori  RC,  Pagacz  J,  Frost  GB,  Efimova  EV, et al. (2022) Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization. Cell Chem Biol 29: 1517–1531.e7.
• 131   Song  H,  Chen  X,  Jiao  Q,  Qiu  Z,  Shen  C, et al. (2021) HIF-1α-Mediated telomerase reverse transcriptase activation inducing autophagy through mammalian target of rapamycin promotes papillary thyroid carcinoma progression during hypoxia stress. Thyroid 31: 233–246.
• 132   Cancer Genome Atlas Research  Network (2014) Integrated genomic characterization of papillary thyroid carcinoma. Cell 159: 676–690.
• 133   Wang  N,  Xu  D,  Sofiadis  A,  Hoog  A,  Vukojevic  V, et al. (2014) Telomerase-dependent and independent telomere maintenance and its clinical implications in medullary thyroid carcinoma. J Clin Endocrinol Metab 99: E1571–E1579.
• 134   Chindris  AM,  Casler  JD,  Bernet  VJ,  Rivera  M,  Thomas  C, et al. (2015) Clinical and molecular features of Hurthle cell carcinoma of the thyroid. J Clin Endocrinol Metab 100: 55–62.
• 135   Qasem  E,  Murugan  AK,  Al-Hindi  H,  Xing  M,  Almohanna  M, et al. (2015) TERT promoter mutations in thyroid cancer: a report from a Middle Eastern population. Endocr Relat Cancer 22: 901–908.
• 136   Kim  TH,  Kim  YE,  Ahn  S,  Kim  JY,  Ki  CS, et al. (2016) TERT promoter mutations and long-term survival in patients with thyroid cancer. Endocr Relat Cancer 23: 813–823.
• 137   Liu  R,  Bishop  J,  Zhu  G,  Zhang  T,  Ladenson  PW, et al. (2017) Mortality risk stratification by combining BRAF V600E and TERT promoter mutations in papillary thyroid cancer: genetic duet of BRAF and TERT promoter mutations in thyroid cancer mortality. JAMA Oncol 3: 202–208.
• 138   Ibrahimpasic  T,  Xu  B,  Landa  I,  Dogan  S,  Middha  S, et al. (2017) Genomic alterations in fatal forms of non-anaplastic thyroid cancer: identification of MED12 and RBM10 as novel thyroid cancer genes associated with tumor virulence. Clin Cancer Res 23: 5970–5980.
• 139   Liang  J,  Cai  W,  Feng  D,  Teng  H,  Mao  F, et al. (2018) Genetic landscape of papillary thyroid carcinoma in the Chinese population. J Pathol 244: 215–226.
• 140   de la Fouchardiere  C,  Decaussin-Petrucci  M,  Berthiller  J,  Descotes  F,  Lopez  J, et al. (2018) Predictive factors of outcome in poorly differentiated thyroid carcinomas. Eur J Cancer 92: 40–47.
• 141   Trybek  T,  Walczyk  A,  Gasior-Perczak  D,  Palyga  I,  Mikina  E, et al. (2019) Impact of BRAF V600E and TERT promoter mutations on response to therapy in papillary thyroid cancer. Endocrinology 160: 2328–2338.
• 142   Vianello  F,  Censi  S,  Watutantrige-Fernando  S,  Barollo  S,  Zhu  YH, et al. (2021) The role of the size in thyroid cancer risk stratification. Sci Rep 11: 7303.