Background
Soft tissue sarcomas (STS) are a highly heterogeneous group of malignant tumors of mesenchymal origin represented by voluntary muscles, fat, and fibrous tissue and their vessels and by convention the peripheral nervous system [
1]. STS are relatively rare and constitute approximately 1–2% of all human cancers, but incidence dramatically increases with age [
1,
2].
Since most patients with STS present with a painless swelling, a delayed diagnosis is common, often with local or distant metastatic spread at the time of diagnosis [
2]. The treatment of choice depends on the individual tumor type, grading and staging status. Surgery, among others, is a key element of therapy in sarcomas of adults with the aim of microscopically tumor-negative margins for optimal local control [
3]. However, standardized treatment might be insufficient. Under these circumstances, advance in personalized treatment strategies might become important with the goal to individual tumor-targeted therapies.
That is why the biology of STS has intensively been investigated over the last decades with a dramatic increase of knowledge about genetic alterations [
4] including aberrant DNA methylation [
5]. In general, sarcomas can be classified into two genetic groups: i. sarcomas with specific chromosomal rearrangements on a background of relatively few other chromosomal changes, and ii. sarcomas without specific alterations on a complex background of numerous chromosomal changes [
6]. Specific genetic alterations are not only of diagnostic significance, but also might become relevant for tumor-targeted therapies.
Telomere maintenance is an important step during tumorigenesis and confers unlimited proliferative capacity to cancer cells [
7]. In principal, two mechanisms can be involved in telomere maintenance: a telomerase dependent mechanism or a non-telomerase dependent mechanism also referred to as Alternative Lengthening of Telomeres (ALT) [
7]. The ribonucleoprotein complex telomerase provides the physiological mechanism that maintains telomere length by adding repetitive hexanucleotide repeats with the sequence 5′-TTAGGG-3′ to telomeres. Reactivation of telomerase has been observed in the majority of human cancers [
8]. In this context, telomerase reverse transcriptase (
TERT) serves as the catalytic subunit of the telomerase complex and has been shown to contribute to the immortalization of cancer cells [
7]. However, the underlying mechanism of
TERT reactivation in cancer cells was an unresolved issue [
9].
Recently, highly recurrent somatic mutations in the promoter region of the
TERT gene have been detected [
10]. The most frequent mutations were a single cytosine exchange to thymine at chromosome 5 base position 1,295,228 (C228T) or less frequently at base position 1,295,250 (C250T) (-124 and -146 bp from ATG start site, respectively). These
TERT mutations lead to a new binding motif for E-twenty six/ternary complex factors (Ets/TCF) transcription factors and results in an up to 4-fold increase of
TERT promoter activity in reporter gene assays [
11,
12].
First described in melanomas [
11,
12],
TERT promoter mutations have subsequently been found in many other human cancer types, with highest frequencies in subtypes of CNS tumors, in a number of malignancies of epithelial origin including bladder carcinomas, thyroid carcinomas, and hepatocellular carcinomas, and in atypical fibroxanthomas and in dermal pleomorphic sarcomas [
13‐
26]. Accordingly,
TERT promoter mutations belong to the most common somatic genetic lesions in human cancers.
A study by Killela et al. investigated a broad range of human cancers for
TERT promoter mutations, including soft tissue sarcomas [
16]. However, the case number of single STS entities was limited and a number of subtypes were not comprised.
Therefore, the present study was conducted to investigate the prevalence of TERT promoter mutations in a comprehensive series of 341 soft tissue tumors comprised of 16 types including rare entities and in 16 cell lines of seven sarcoma types. Further, we looked for associations, if any, with clinicopathological parameters.
Discussion
Telomere maintenance mechanisms represent a pivotal cornerstone in the development and sustainment of cancer. The recently described mutations in the promoter region of TERT provide new evidence for the important role of telomerase reactivation in human cancers. The overall prevalence of TERT promoter hotspot mutations was low in the comprehensive series of soft tissue sarcomas examined in this study (36/341; 10.5%). However, the prevalence strongly varied by sarcoma type.
The by far highest mutation rate was found in MLS (29/39; 74%), which represents the most prevalent mutation identified in this sarcoma entity to date, and which corroborates data obtained in a recent study on a smaller series of MLS [
16]. In MLS, increased
TERT transcription [
27‐
29] and telomerase reactivity [
28] have been described previously. Costa et al. found telomerase reactivation in 69% of MLS with an additional round cell component (high grade) [
28], which is overlapping with the overall
TERT promoter mutation frequency of 74% (29/39) in our series of MLS. However, in pure MLS without the round cell phenotype (corresponding to low grade), they found telomerase reactivation only in 39% of cases [
28]. Likewise, Schneider-Stock et al. detected telomerase activity in 30% of MLS, but elevated
TERT mRNA levels in a much higher proportion of cases [
27,
29,
30]. Furthermore, intratumoral heterogeneity of
TERT expression and telomerase activity has been observed in sarcomas, in particular in liposarcomas [
31]. Thus,
TERT mRNA levels are not stringently correlated with telomerase enzyme activity. This might be explained by sufficient regulatory mechanisms of the enzymatic function of telomerase, which still have to be functional in some tumors.
Indeed, regulatory mechanisms of telomerase have already been described at the transcriptional and post-translational level. At the transcriptional level, alternative splicing of
TERT mRNA itself might not only lead to TERT variants with impaired catalytic functions [
32], but also to a variant that acts in a dominant-negative manner on telomerase activity [
33]. Furthermore, post-translational modifications of the TERT protein through phosphorylation or ubiquitination have been shown to affect the catalytic activity and stability of TERT [
34].
Anyhow, our data suggest that mutation of the
TERT promoter causes telomerase reactivation in MLS and thereby most probably provides unlimited proliferative potential. This assumption is also underpinned by a reporter gene assay of the two most common mutation variants within the promoter region of
TERT, namely C228T and C250T, which were shown to lead to an augmented expression of
TERT[
12]. Further, the high prevalence of
TERT promoter mutations not only in MLS round cell variants but also in MLS with a pure myxoid phenotype, and this irrespective of tumor grading, implies that these mutations act rather as driver than passenger mutations.
TERT promoter mutations might also have a diagnostic impact in myxoid sarcomas. Mutations were found neither in dedifferentiated liposarcomasa (DDLS), nor in pleomorphic liposarcomas (PLS), which presented myxoid areas in many cases, and were also not detectable in our series of myxofibrosarcomas, extraskeletal myxoid chondrosarcomas, dermatofibrosarcomata protuberans, and low-grade fibromyxoid sarcomas.
The absence of
TERT promoter hotspot mutations in our series of DDLS and PLS is in line with previous studies, which largely observed deficient telomerase activity in high-grade liposarcomas. Instead, high-grade liposarcomas often use the ALT mechanism [
28,
35,
36]. ALT overcomes telomere attrition through homologous recombination of telomeric DNA and characteristically presents with a pattern of telomere lengths that range from very short to abnormally long. This telomere pattern is clearly different compared to tumors with telomerase reactivation, where telomere length is found almost equal [
36].It has been shown that ALT-positive liposarcomas have a notably worse outcome, and may imply a more favorable prognosis for
TERT promoter mutated liposarcomas [
28,
37,
38]. However, differences in patients outcome might be dedicated to the fact that telomere maintenance via ALT is more often applied by tumors with complex karyotypes or with a higher level of genomic instability [
39,
40], whereas sarcomas characterized by type specific translocations rather use telomerase reactivation for telomere maintenance [
39,
41]. According to our data, this concept holds true for the group of liposarcomas. MLS are characterized by a translocation that fuses the
DDIT3 (
CHOP) gene on chromosome 12q13 with the
FUS (
TLS) gene on chromosome 16p11 in approximately 90% of cases, or the
DDIT3 (
CHOP) with the
EWSR1 on chromosome 22q12 in the remaining cases [
42]. By contrast, DDLS typically have complex karyotypic aberrations with amplification of the chromosome 12 subregion q13-15, which includes the
murine double minutes (
MDM2) and
cyclin dependent kinase-4 (
CDK4) genes among others [
43,
44]. PLS are characterized by highly complex karyotypes [
45]. The highest prevalence of ALT has been observed in DDLS and PLS, which typically have an aggressive biological behavior [
28,
37]. However,
TERT promoter mutated MLS may undergo malignant progression to the round cell variant and then present with a similar biological behavior like ALT-positive PLS [
46]. Another fact that challenges this concept is that patients suffering from ALT-positive glioblastoma have a more favorable clinical course compared to ALT-negative counterparts [
47,
48]. Thus, the unfavorable prognosis in ALT-positive liposarcomas is probably derived from the mutational signature in these tumors rather than dependent on the mechanism of telomere maintenance, and thus may considerably differ between different tumor entities.
The second most common rate of
TERT promoter mutations was observed in SFT with a frequency of 13%, which is concordant to data on a smaller series of SFTs [
16]. However,
TERT promoter mutation might be dependent on the anatomic site of presentation, since cranial SFTs and hemangiopericytomas, which are now considered to belong to the SFT family from a genetic perspective [
49], have a slightly higher mutation frequency (11/43; 26%) [
17].
In MPNSTs,
TERT promoter mutations were found in a small fraction of tumors (2/35; 6%), which is slightly below the mutation frequency previously reported (2/12; 17%) [
17]. These data might suggest a minor significance in this tumor entity. On the other hand, one out of three MPNST cell lines was revealed with a
TERT promoter mutation, which supports the assumption that telomerase reactivation by
TERT promoter mutations might contribute to immortalization of at least a small proportion of MPNSTs. Interestingly, a previous study that focused on telomerase activity in MPNSTs found telomerase reactivation in 14 of 23 (61%) MPNSTs [
50]. Compared with histological grade, telomerase activity was completely restricted to high grade MPNSTs (14/17; 82%) in that study. Indeed, the two MPNSTs with
TERT promoter mutation described here presented with typical histological features of high-grade MPNSTs [
51]. Moreover, in another study on 57 MPNST samples telomerase activity proved to be significantly associated with disease-specific mortality during 5 years of follow-up [
52].
Another notable observation is the sporadic occurrence of
TERT promoter mutations in SSs. This tumor typically applies telomerase reactivation for telomere maintenance [
53], which is in concordance with our own observations (data not shown). However, like in MPNSTs,
TERT promoter hotspot mutations just play a minor role in SSs with merely a single mutated case in our series (1/25; 4%). Thus, the low mutation frequencies in MPNSTs and SSs suggest that a so far unknown mechanism beside the
TERT promoter hotspot mutations may exist that provides telomerase reactivation.
Explanations for telomerase maintenance get complicated by the observation that a considerable fraction of STS do neither apply telomerase activation nor the ALT mechanism that is so far known, or even may be equipped with both mechanisms [
7,
36]. Further studies concerning molecular alterations in STS will in particular draw more attention to the non-coding genomic regions and hopefully elucidate the remaining unanswered questions, which mechanisms these tumors exploit to prevent telomere attrition.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CK and MR contributed equally to this work. CK, MR, WH, EW, TS, GE, PS, AvD and GM performed data analyses. WH, EW and GM carried out the histological review of cases. CK, MR, NW and RP performed molecular analyses. AU, ERK, BL, IA, PS and GM collected cases. CK and GM conceived and designed the study, and prepared the initial manuscript. GM supervised the project. All authors contributed to the final manuscript. All authors read and approved the final manuscript.