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A Pan-Cancer Study of Somatic TERT Promoter Mutations and Amplification in 30,773 Tumors Profiled by Clinical Genomic Sequencing
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Sounak Gupta, * Chad M. Vanderbilt, * Yun-Te Lin, * Jamal K. Benhamida, * Achim A. Jungbluth, * Satshil Rana,* Amir Momeni-Boroujeni,* Jason C. Chang, * Tiffany Mcfarlane,* Paulo Salazar,* Kerry Mullaney,* Sumit Middha,* Ahmet Zehir,* Anuradha Gopalan, * Tejus A. Bale, * Ian Ganly, Maria E. Arcila, * Ryma Benayed,* Michael F. Berger,* Marc Ladanyi,* and Snjezana Dogan*
From the Departments of Pathology* and Surgery,1 Memorial Sloan Kettering Cancer Center, New York, New York
Accepted for publication November 10, 2020. Address correspondence to Snjezana Dogan, M.D., Molec- ular Genetic Pathology Fellow- ship Program, Department of Pathology, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10065. E-mail: dogans@ mskcc.org.
TERT gene promoter mutations are known in multiple cancer types. Other TERT alterations remain poorly characterized. Sequencing data from 30,773 tumors analyzed by a hybridization capture next- generation sequencing assay (Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets) were analyzed for the presence of TERT alterations. Promoter rear- rangements (500 bases upstream of the transcriptional start site), hypermethylation (n = 57), and gene expression (n = 155) were evaluated for a subset of cases. Mutually exclusive and recurrent promoter mutations were identified at three hot spots upstream of the transcriptional start site in 11.3% of cases (-124: 74%; - 146: 24%; and -138: < 2%). Mutually exclusive amplification events were identified in another 2.3% of cases, whereas mutually exclusive rearrangements proximal to the TERT gene were seen in 24 cases. The highest incidence of TERT promoter mutations was seen in cutaneous melanoma (82%), whereas amplification events significantly outnumbered promoter muta- tions in well-differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%). Gene expression analysis suggests that the highest levels of gene expression are seen in cases with amplifications and rearrangements. Hypermethylation events upstream of the TERT coding sequence were not mutually exclusive with known pathogenic alterations. Studies aimed at defining the prevalence and prognostic impact of TERT alterations should incorporate other pathogenic TERT alterations as these may impact telomerase function. (J Mol Diagn 2021, 23: 253-263; https://doi.org/10.1016/j.jmoldx.2020.11.003)
Human telomeres are composed of telomeric TTAGGG DNA repeats that are protected by the shelterin complex and the telomerase complex.1,2 Unlike stem cells, in most differentiated cells, silencing of telomerase reverse tran- scriptase (TERT) occurs over time with consequent telo- meric shortening. This leads to activation of DNA damage response signaling and replicative arrest. An early event in many neoplasms involves bypassing this replicative arrest through telomerase reactivation, thereby increasing both cancer cell viability and genomic instability. The most common mechanism of telomerase reactivation across several cancer types involves TERT reactivation through hot spot promoter mutations, which generates a de novo binding
site for activating erythroblast transformation-specific (ETS) family transcription factors.24 This leads to increased recruitment of multimeric GA-binding protein transcription factors to the mutant TERT promoter sequence and tran- scriptional up-regulation of TERT.2,3,5
In addition to promoter mutations, the spectrum of acti- vating TERT alterations includes genomic amplifications as
Supported in part through NIH/National Cancer Institute Cancer Center (NCI) under the MSK Cander Center Support Grant/Core Grant P30CA008748.
S.G. and C.M.V. contributed equally to this work. Disclosures: None declared.
well as structural alterations at the TERT locus or hyper- methylation events upstream of the TERT transcriptional start site.6-10 The Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK- IMPACT) assay used in this study is designed to detect TERT promoter mutations and genomic amplifications. Although a few structural variants were detected within a short 500-bp sequence immediately upstream of the tran- scriptional start site, the vast majority of these rearrange- ments (which have primarily been reported in neuroblastomas) occur over a 50-kb region proximal to the TERT gene.7-9 This assay is therefore not designed to detect these alterations when they occur further upstream of the transcription start site. Finally, hypermethylation at specific CpG islands upstream of the transcriptional start site has been correlated with increased TERT gene expression, and this assay does not document methylation status at these sites.6,10,11 Methylation status was determined for a smaller subset of cases using an alternate assay.
Relatively few large-scale pan-cancer studies have stud- ied TERT alterations across tumor types.2,12,13 Herein, we present pan-cancer data pertaining to both promoter muta- tions and amplification events across 30,773 specimens that were profiled as part of an institutional clinical sequencing cohort. This data set is unique as TERT promoter mutations are often not evaluated in whole exome sequencing assays, whereas TERT amplification events are rarely documented in large pan-cancer data sets.14 In addition, a limited number of cases in this study have been profiled for rarer alterations, such as structural variants and hypermethylation events upstream of the TERT transcription start site, which have been shown to up-regulate TERT expression, as well as relative gene expression status.
Materials and Methods
Case Selection
This study was approved by the institutional review board and involved analysis of molecular profiling data of multiple solid tumors profiled by a next-generation sequencing-based assay (MSK-IMPACT), as part of an institutional clinical cancer genomics initiative.14-16 DNA sequencing results for 30,773 tumor samples obtained from formalin-fixed, paraffin-embedded tissue, performed in a Clinical Laboratory Improvement Amendments-approved setting, were analyzed for TERT alterations.
Next-Generation Sequencing: MSK-IMPACT
Details of the MSK-IMPACT assay have been previously reported.14-16 In brief, this assay involves paired analysis of tumor and normal specimens to filter out germ-line variants. Specifically, hybridization capture-based library preparation is followed by deep sequencing of select noncoding regions and 6614 protein-coding exons of 468 genes. Noncoding
sequences include the TERT promoter (extending to 500 bp upstream of the transcriptional start site), intronic sequences of commonly rearranged genes, microsatellite sites, and several single-nucleotide polymorphisms. In all, the capture probes target approximately 1.5 megabases of the human genome. Accurate genome-wide copy number assessment is facilitated by homogeneous distribution of single-nucleotide polymorphism tiling probes across the genome. On the basis of previously reported criteria, amplifications were defined as a fold change ≥2.0.17-20 Rearrangements involving the TERT promoter (500-bp sequence immediately upstream of the transcriptional start site) were identified on the basis of previously published criteria, which included the following: five paired or split reads, a mapping quality of 20, and a length >500-bp sequence.15 This assay is currently approved by the US Food and Drug Administration as a class II in vitro diagnostic test.
TERT Gene Expression Analysis
Details pertaining to RNA extraction and the MSK-Fusion assay have been previously reported.21,22 Eight gene- specific primers were designed by ArcherDx (Boulder, CO) and added to the MSK-Fusion panel to specifically detect TERT expression levels. The total number of unique RNA reads for TERT, obtained using all eight gene-specific primers, were normalized to the total number of unique RNA reads for five separate housekeeping genes interro- gated as part of the assay. The relative TERT gene expres- sion status was evaluated for 155 cases that were profiled using MSK-IMPACT from available archived formalin- fixed, paraffin-embedded tissue. Broad categories of tumor types evaluated included colorectal adenocarcinoma (n = 35), lung adenocarcinoma (n = 25), sarcoma (n = 20), gliomas (n = 19), breast carcinoma (n = 14), melanoma (n = 12), pancreaticobiliary cancer (n = 10), salivary cancer (n = 4), thyroid carcinoma (n = 4), carcinoma of unknown primary (n = 4), and miscellaneous tumor types (n = 8).
TERT Promoter Methylation Analysis
TERT promoter hypermethylation status was detected via bisulfite conversion followed by methylation array for 57 cases profiled by MSK-IMPACT, as previously described.23 Herein, methylation status of three CpG sites upstream of the TERT transcription start site was evaluated. This included the cg11625005 CpG site, where hypermethylation has been previously reported to be associated with high- grade tumors and increased TERT gene expression. 6,10,11
Statistical Analysis
All statistical tests were two sided, and P < 0.05 was considered statistically significant.
Results
TERT Promoter Mutation and Amplification
A hybrid capture library preparation strategy was used to sequence the TERT promoter region, including 500 bases upstream of the transcriptional start site. The mean depth of coverage for the promoter region across all 30,773 speci- mens that were profiled was 425x, with an interquartile range of 240x to 551x (Figure 1A). In all, 4205 of these specimens (13.6%) harbored either a TERT promoter
mutation or an amplification. Of note, TERT promoter mu- tations (11.3%) and amplifications (2.3%) were mutually exclusive, with the promoter mutations occurring 4.9 times as frequently compared with amplification events (Figure 1B).
Although TERT promoter mutations showed a wide spatial distribution, consistent with our prior studies, three recurring hot spots that were mutually exclusive were identified.14 Relative to the transcription start site, hot spot promoter alterations at position -124 were the most
A
1250-
B
8000-
:
1000-
TERT Amplification (n = 715; 2.3%)
6000
.
Coverage
750-
4000-
TERT Promoter Mutation (n = 3490; 11.3%)
500-
250
2000
No TERT Amplification/ Promoter Mutation (n = 26,568; 86.3%)
0-
0-
TERT
TERT
Gene
C
CCTCCGG > CTTCCGG n =2374
3000
CCTCCGG > CATCCGG n=30 CCTCCGG > AATCCGG n= 44
-124C>T/C>A / CC>AA
Patients, n
1000
CCTCCCG > CCTTCCG n = 784 CCTCCCG > TCTTCTT n=6
-146C>T/CCTC>TCTT
TCCCCGG > TTTCCGG n =57
-138CC>TT
50
3
9
9
9
8
9
99919
TERT SUTR
TERT Coding Region
-350
-146
-124
-58
D
-138
100
Patients, %
75
50
25
0
Melanoma
Urothelial Carcinoma
Basal Cell Carcinoma
Cutaneous Squamous Cell Carcinoma
Carcinoma of Unknown Primary
Cholangiocarcinoma
Lung Adenocarcinoma
Papillary Thyroid Carcinoma
% of Patients
A
B
100
100
Patients, %
☐ No Amplification/ Promoter Mutation
75
75
Promoter Mutations
Patients, %
No Amplification/ ☐ Promoter Mutation
50
50
Promoter Mutations
25
Amplifications ☐
25
☐
Amplifications
0
0
Cutaneous Squamous Cell Carcinoma (n = 93)
Basal Cell Carcinoma (n = 33)
Merkel Cell Carcinoma (n = 90)
Cutaneous Melanoma (n = 526)
Mucosal Melanoma (n = 135)
Ocular Melanoma (n = 14)
% of Patients
% of Patients
C
100
D
100
Patients, %
80
Patients, %
75
60
50
40
20
25
0
0
Low- Grade
Glioma (TERT+; IDH WT)
High- Grade Glioma (TERT+; IDH WT)
Low- Grade Glioma (TERT+; IDH Mt)
High- Grade Glioma (TERT+; IDH Mt)
(Upper Tract) Urothelial Carcinoma (n = 151)
(Urinary Bladder) Urothelial Carcinoma (n = 626)
(Urethral) Urothelial Carcinoma (n = 19)
Renal Cell Carcinoma (n = 682)
Prostate Cancer (n = 1906)
Testicular Germ Cell Tumor (n = 357)
(n = 96)
(n = 855)
(n = 161)
(n = 264)
Amplifications
☐ Promoter Mutations
☐ No Amplification/ Promoter Mutation
% of Patients
% of Patients
E
100
Patients, %
75
☐ No Amplification/ Promoter Mutation
50
Promoter Mutations
25
Amplifications ☐
0
Anaplastic Thyroid Carcinoma (n = 70)
Papillary Thyroid Carcinoma (Tall Cell Variant) (n = 127)
Poorly Differentiated Thyroid Carcinoma (n = 116)
Follicular Thyroid Carcinoma (n = 11)
Hurthle Cell Carcinoma (n = 42)
* Papillary Thyroid Carcinoma (n = 104)
Medullary Thyroid Carcinoma (n = 41)
F
% of Patients Promoter Mutations
Amplifications
No Amplification/ Promoter Mutation
100
Patients, %
75
50
25
0
Myxoid Liposarcoma (n = 39)
Hepatocellular Carcinoma (n = 188)
Granulosa Cell Tumor (n = 27)
Fibrolamellar Carcinoma (n = 31)
Adrenocortical Carcinoma (n = 66)
Well/ Dedifferentiated Liposarcoma (n = 170)
Adenoid Cystic Carcinoma (n = 156)
Lung Adenocarcinoma (n = 3662)
Colorectal Adenocarcinoma (n = 2749)
Ovarian Cancer (n = 820)
Neuroblastoma (n = 205)
Endometrial Cancer (n = 1068)
Amplifications
Promoter Mutations
☐ No Amplification/ Promoter Mutation
common (n = 2448; 74%), followed by those at positions -146 (n = 790; 24%) and -138 (n = 57; < 2%) (Figure 1C). Although TERT promoter mutations at the -138 position were seen in varied tumor types, they predominantly occurred
in melanoma (75.8% of cases) (Figure 1D). Consistent with prior reports, G-to-A substitutions at these sites led to the formation of presumptive ETS transcription factor binding sites, including at position -138.14
Relative Frequency of TERT Alterations in Multiple Tumor Types
Solid tumor types with high rates of TERT alterations (in combined primary and metastatic tumors) have been high- lighted (Figure 2). Cutaneous neoplasia, such as squamous cell carcinoma, basal cell carcinoma, and cutaneous mela- noma, primarily harbored TERT hot spot promoter muta- tions in association with a UV-induced mutational signature (ie, predominant G-to-A and C-to-T mutations), whereas amplifications were relatively rare (Figure 2, A and B). In contrast, similar alterations were infrequently identified in Merkel cell carcinoma and mucosal/ocular melanoma. Consistent with prior reports, TERT hot spot mutations in central nervous system glial neoplasia predominantly occurred in an IDH1 wild-type setting in high-grade gliomas (84% of IDH1 wild-type cases) compared with low-grade gliomas, where they tended to occur in association with IDH1 mutations (56% of IDHI mutant cases) (Figure 2C).24-26 Among genitourinary malignancies, the highest incidence was seen for urothelial carcinoma, with a relatively lower frequency observed for upper tract disease (53% versus 74%) (Figure 2D). As previously reported, TERT promoter mutations showed a strong association with the underlying subtype in thyroid carcinoma (Figure 2E).13,27-33 No alterations were identified in med- ullary thyroid carcinoma, and up to 77% of anaplastic thy- roid carcinomas harbored these alterations. Interestingly, although a lower frequency was seen for papillary thyroid carcinoma (including classic and follicular variants; 38%), the aggressive tall cell variant showed a much higher fre- quency (68%). As expected, other tumor types with a high frequency of TERT promoter mutations included myxoid liposarcoma, hepatocellular carcinoma, and granulosa cell tumors (Figure 2F). 34-36 In contrast, tumor types where TERT amplification was the prevalent alteration included
well-differentiated/dedifferentiated liposarcoma (14.1%) and adrenocortical carcinoma (13.6%) (Figure 2F). 37,38
Of note, high rates of TERT alterations were documented in the primary tumors themselves for aggressive variants of thyroid cancer, such as poorly differentiated (n = 73; 52%), anaplastic (n = 55; 69%), and tall cell variant of papillary thyroid cancer (n = 43; 74%), as is depicted in Figure 3. In contrast, these alterations were predominantly documented in metastatic/recurrent tumors for classic well-differentiated papillary thyroid cancer (n = 58; 52%) and Hurthle cell carcinoma (n = 26; 54%).
TERT Promoter Rearrangements
Prior studies have identified recurrent genomic rearrange- ments at the TERT locus, upstream of the TERT coding sequence, primarily in high-risk neuroblastoma.7,8 These cases showed substantial induction of TERT gene expres- sion and downstream increases in telomerase activity, likely secondary to the juxtaposition of strong enhancer elements adjacent to the TERT coding sequence.7,8 Although subse- quent studies have confirmed the presence of TERT pro- moter rearrangements, these remain challenging to identify as these rearrangements often occur over a 50-kb region proximal of the TERT gene.9,11,39,40 Although the MSK- IMPACT assay only tiles for 500 bases of this 50-kb re- gion, 24 cases occurring in multiple tumor types were found to harbor similar rearrangements upstream of the TERT coding sequence (Figure 4). No recurrent break point or partner gene was identified for these cases. In addition, these events were mutually exclusive with TERT hot spot pro- moter mutations/genomic amplifications.
Relative TERT Gene Expression
Herein, 155 specimens of archived formalin-fixed, paraffin- embedded specimens that were initially evaluated using
Primary
Metastasis
100
90
**
80
Patients, %
70
60
50
40
30
20
10
0
Renal Cell Carcinoma
Merkel Cell Carcinoma
Mucosal Melanoma
Adrenocortical Carcinoma
Papillary Thyroid Carcinoma
Hurthle Cell Carcinoma
Granulosa Cell Tumor
Poorly Differentiated Thyroid Carcinoma
Follicular Thyroid Carcinoma
Anaplastic Thyroid Carcinoma
Cutaneous Melanoma
Urothelial Carcinoma
Papillary Thyroid Carcinoma (Tall Cell)
Myxoid Liposarcoma
Primary
Metastasis
MSK-IMPACT were retrieved and further interrogated for TERT mRNA expression using the MSK-Fusion assay. Unique TERT-specific RNA reads were normalized to the expression of multiple housekeeping genes and quantified. This included cases that lacked TERT alterations or harbored amplifications, hot spot promoter mutations, or rearrange- ments. Interestingly, cases with genomic amplifications and promoter rearrangements showed statistically significant increases in TERT gene expression, in contrast to cases with hot spot promoter mutations (Figure 5).
Although multiple studies have attempted to demonstrate TERT protein expression using immunohistochemistry, this has historically been controversial. Such efforts have been hampered by poor reproducibility, unexpected patterns of subcellular localization, as well as documented cross- reactivity with other proteins.41,42 Validation of such an immunohistochemical assay would require establishing a baseline status for all cases that was inclusive of not only promoter mutations, but also amplifications, upstream rearrangements, and methylation events to correlate protein expression with underlying tumor biology. Although mul- tiple antibodies were to be validated, the validation failed because of a lack of consistency with the underlying pre- diction of TERT expression status and unexpected patterns of protein localization (data not shown).
TERT Promoter Hypermethylation Status
Recent studies have suggested that hypermethylation up- stream of the TERT transcriptional start site is required for TERT gene expression and corresponding telomerase ac- tivity in neoplastic cells compared with nonneoplastic cells.6,9-11 At least one study has suggested that these hypermethylation events are not mutually exclusive with either TERT promoter mutations or rearrangements.1 11
Adrenocortical Carcinoma (n = 1)
Lung Adenocarcinoma (n = 1)
Carcinoma of Unknown Primary (n = 2)
Sarcoma (n = 4)
Prostate Adenocarcinoma (n = 2)
Urothelial Carcinoma (n = 3)
Breast Cancer (n = 2)
Renal Cell Carcinoma (n = 3)
Mesothelioma (n = 2)
Melanoma (n = 2)
High-Grade Glioma (n = 2)
Although cases with hypermethylation were not evaluated for relative gene expression, 57 cases of high-grade glioma profiled by MSK-IMPACT were further evaluated for methylation status upstream of the TERT gene. This was performed to determine if hypermethylation events were mutually exclusive with activating alterations, such as hot spot promoter mutations and amplifications. Our results suggest that these are not mutually exclusive events as 37 of 42 (88%) of cases with promoter mutations also exhibited hypermethylation upstream of the transcriptional start site (Figure 6).
Discussion
Telomerase reactivation in cancer is often driven by TERT expression, and the predominant underlying alteration in- volves promoter mutations that generate a de novo binding site for activating ETS family transcription factors that lead to transcriptional up-regulation.15 Other alterations that can up-regulate TERT expression include genomic amplifi- cation and rearrangements or hypermethylation events up- stream of the TERT transcriptional start site.
The mean coverage for the TERT promoter region across all 30,773 specimens evaluated by MSK-IMPACT was 425x and revealed the presence of TERT alterations in 13.7% of all cases. Promoter mutations were found to be 4.9 times more prevalent compared with amplification events. Although promoter mutations were distributed along the length of the entire promoter region that was sequenced, consistent with our prior report, three recurrent and mutually exclusive hot spots were identified.14 Among the hot spot mutations, the canonical alterations at positions -124 and -146 bases relative to the transcription start site accounted for 74% and 24% of cases, respectively. An additional noncanonical hot spot at position -138 accounted for <2% of cases, and all these alterations are predicted to generate de novo ETS transcription factor binding sites. 4 Therefore, this latter category of promoter mutations (-138) might be missed in a small subset of cases in targeted assays that interrogate/report only the canonical hot spot alterations (-124 and -146). Similarly, amplification events can be missed in a much larger subset of cases. This is particularly relevant for tumor types where amplification events signif- icantly outnumber promoter mutations, such as in well- differentiated/dedifferentiated liposarcoma (14.1% versus 2.4%) and adrenocortical carcinoma (13.6% versus 4.5%).37,38,43
TERT alterations were found to be enriched in specific tumor types, and a subset of these has been highlighted with the caveat that the institutional (tertiary referral center) clinical sequencing cohort presented herein has a bias to- ward patients who present with high stage and aggressive disease.
Some relevant associations include cutaneous mela- nomas, which showed a high rate of TERT alterations (85%)
100
Relative Expression Units: Log Scale
**
*
10
T
T
1
No TERT Alteration (MSK-IMPACT; n = 100)
TERT Promoter Mutation (n = 27)
Gain/Amplification (n = 21)
Promoter Rearrangement (n = 7)
in a background of a UV-induced mutational signature, as opposed to both mucosal (10%) and ocular melanoma (0%), and these trends and frequencies are consistent with what has been previously reported in the literature.2,13,44 A similar high incidence of TERT alterations was seen in sun- exposed cutaneous squamous cell (72%) and basal cell carcinomas (82%).3
Prior studies have shown that TERT promoter mutations in high-grade gliomas occur infrequently in the background of IDH1 mutations (<3%) as opposed to low-grade gliomas (34%).26 Our results support this trend as the vast majority of TERT alterations in high-grade gliomas occurred in the IDHI wild-type setting (n = 856; 84%) and, similarly, a large number of TERT alterations in low-grade gliomas occurred in the IDHI mutant setting (n = 161; 56%).
Among genitourinary cancers, TERT alterations had an exceedingly low prevalence in prostatic adenocarcinoma
Hypermethylation+
Hypermethylation-
100
Patients, %
75
50
25
0
No TERT Alteration (MSK-IMPACT; n = 14)
TERT Promoter Mutation (n = 42)
TERT Amplification (n = 1)
Hypermethylation+
Hypermethylation-
and testicular germ cell tumors (<0.5%), followed by a low incidence in renal cell carcinoma (5.7%), as has been pre- viously reported.2,14,45 The highest incidence was seen in urothelial carcinoma (overall: 73%). The overall frequency is consistent with published reports that suggest that on the basis of a high incidence, and the observation that TERT alterations may be an early event in urothelial carcinoma, the detection of these alterations may be exploited as a potential urothelial cancer screening tool, particularly using urine/cytology specimens.2,12,13,46-48 On the basis of our results, some variables that may confound the results of urothelial cancer screening assays that rely on the detection of TERT promoter mutations include the presence of these alterations in a subset of renal cell carcinoma (false posi- tives), whereas the absence of TERT alterations in a subset of cases or the presence of amplification events (2.6% of cases) may contribute to false negatives. 45,46,48
With regard to thyroid cancer, prior studies have shown absence of TERT promoter mutations in medullary thyroid carcinoma, a relatively low incidence in papillary carcinoma (7% to 25.5%), Hurthle cell carcinoma (13% to 33%), and follicular thyroid carcinoma (14% to 36%), and the highest incidence in poorly differentiated carcinoma (21% to 60%) and anaplastic thyroid carcinoma (13% to 73%); these studies suggest that these alterations are enriched in thyroid carcinomas that exhibit more aggressive behavior.2,13,27-33 In our series of 511 thyroid carcinomas, with many pa- tients presenting with advanced disease, an absence of TERT promoter mutations in medullary thyroid carcinoma was confirmed, followed by progressively increasing incidence in papillary, Hurthle cell, and follicular thyroid carcinomas (39%, 48%, and 55%, respectively) compared with 57% in poorly differentiated and 77% in anaplastic thyroid carci- nomas. TERT amplifications were detected in 2% of cases, which is close to the previously reported incidence of 4.9%.27 Furthermore, consistent with prior reports, our re- sults underline the higher incidence of TERT alterations in the tall cell variant compared with classic well-differentiated
papillary thyroid carcinoma (69% versus 39%).2 Among poorly differentiated, anaplastic and tall cell variants of papillary thyroid carcinoma, there was no significant dif- ference in the frequency of TERT promoter mutations when comparisons were made between primary and metastatic tumors. This finding is consistent with their (more) aggressive biology.30,33 However, among classic well- differentiated papillary thyroid carcinoma and Hurthle cell carcinoma, TERT was more commonly mutated in meta- static/recurrent disease than in primary tumors (classic well- differentiated papillary thyroid carcinoma: 52% versus 30%; and Hurthle cell carcinoma: 54% versus 33%), suggesting that these genetic alterations may identify a relatively more aggressive subset of well-differentiated thyroid carcinomas with a tendency to metastasize/recur. However, these results must be interpreted with caution because of sampling bias (ie, the fact that the vast majority of thyroid cases subjected for testing were from patients with relatively more aggres- sive disease).
Other tumor types that have high reported incidences of TERT alterations include hepatocellular carcinoma (ampli- fications: 10%; and promoter mutations: 44%) and ovarian granulosa cell tumors (22%), whereas myxoid liposarcomas (74%) have the highest reported incidence among soft tissue tumors.34-36 Our results revealed a similar trend, revealing an overall frequency of promoter mutations/amplifications of 59%, 60%, and 82% for hepatocellular carcinoma, granulosa cell tumors, and myxoid liposarcoma, respec- tively. Of note, the frequency of these alterations in ovarian granulosa cell tumors has been reported to be significantly higher among recurrences, and furthermore, their presence has been correlated with poor outcomes.34 In hepatocellular carcinoma, up to 6.4% of cases harbored amplifications of TERT and are likely to be missed if they are evaluated using assays that do not evaluate copy number changes.
With regard to TERT gene expression, although a large number of studies have demonstrated the impact of TERT hot spot promoter mutations on transcriptional up-regulation in vitro, similar studies that have directly evaluated the ef- fect of TERT promoter mutations on gene expression in tumor-derived biospecimens are limited.3 Furthermore, several studies have documented only modest increases in TERT gene expression in both in vitro studies and in tumor specimens.11,46,49 Our results confirm that cases with genomic amplifications and promoter rearrangements showed statistically significant increases in TERT gene expression and highlight the importance of identifying such cases.7-9 In contrast, relative gene expression in cases with promoter mutations did not show a statistically significant increase. This could be secondary to the age and type (archived formalin fixed and paraffin embedded) of spec- imen evaluated, potentially modest increases in gene expression, and/or potentially low sensitivity of the assay used. A limitation of our study involves a lack of correlation of TERT expression with downstream gene expression sig- natures, particularly related to ETS transcription factors. 50,5
This approach was not utilized in the current study as gene expression-specific primers to perform such an analysis were not included in the MSK-Fusion assay. However, future studies may utilize such a strategy when interrogating TERT gene expression status.
Perhaps the most intriguing category of TERT alterations involves structural variants that have been previously re- ported to be present in neuroblastomas.7-9 Specifically, these have been reported in 23% to 31% of high-risk neuroblas- tomas, have exhibited a large structural diversity of TERT promoter rearrangement events, and were frequently clus- tered approximately 50 kb upstream of the TERT transcrip- tional start site.7,8 These events were reported to lead to a juxtapositioning of superenhancer elements and were asso- ciated with a massive transcriptional up-regulation of the TERT gene.7,8 Although reports of such alterations are pre- dominantly restricted to neuroblastomas that show aggressive clinical behavior, 24 such events were identified across diverse tumor types and significantly higher TERT gene expression was reported in seven such cases where archived material was available for downstream analysis, to validate these findings.7-9 In summary, prior studies pertaining to neuroblastoma suggest that TERT promoter rearrangements are associated with high levels of TERT gene expression, and our results confirm these findings.7,8 However, future studies are needed to define the true prevalence of these alterations and their impact on tumor biology.7-9
Recent studies have shown that hypermethylation at specific CpG islands upstream of the TERT transcriptional start site has been associated with increased TERT gene expression, particularly in pediatric brain tumors and in melanoma.6,10,11 As there is a paucity of studies that have evaluated large data sets for such hypermethylation events in the context of other pathogenic alterations, 57 cases with known promoter mutation and copy number status on the basis of profiling by MSK-IMPACT were evaluated. Consistent with the results of a prior study that evaluated a limited number of cases in a similar manner, these events were not mutually exclusive with either amplification events or promoter mutations. However, it is possible that a subset of cases that are considered to be wild type for TERT may in fact harbor epigenetic alterations that have a sig- nificant impact on gene expression.”
Given the high prevalence of TERT alterations in cancer, there have been ongoing efforts to target components of the telomerase holoenzyme.14,52-54 Although such approaches are currently not in clinical use, they represent exciting future cancer therapy strategies. Herein, we provide a snapshot of the landscape of somatic TERT alterations across multiple cancer types. Although canonical hot spot promoter mutations (positions -124 and -146, relative to the transcription start site) are the prevalent pathogenic alteration, our data support the presence of an additional, mutually exclusive, hot spot alteration at position -138.14 Other alterations of note include genomic amplification events and rearrangements proximal to the TERT gene,
which may have a significant impact on gene expression but are likely missed by targeted assays that interrogate only the conventional promoter hot spot mutations. In addition, these data suggest that upstream hypermethylation events, which have been reported to be associated with increased gene expression, are not mutually exclusive with known acti- vating TERT alterations, and additional studies are needed to further define their role across varied cancer types. In summary, studies aimed at defining the prognostic impact of TERT alterations should attempt to incorporate other path- ogenic alterations, including amplifications, upstream rear- rangements, and hypermethylation events, as these might significantly impact telomerase function.
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