With a worldwide estimated age-standardized incidence rate of 4.0 per 100,000 and an estimated number of new cases in 2018 of 354,864, oral cavity squamous cell carcinoma (OCSCC) is the most common carcinoma developing from the epithelial lining of the upper aero-digestive tract (UADT), thus representing an important burden on health care (1).

Based on the histopathological stage, OCSCC can exhibit an unpredictable behaviour with a fraction of patients with early-stage cancer suffering from poor prognosis (2). Patients curatively treated for OCSCC have indeed a high propensity to develop both recurrences and second field tumours (3). Thus, despite recent improvements in the management strategies of OCSCC, improvements in outcomes have been modest (4).

High-risk human papillomaviruses (HPVs), responsible for more than 50% of oropharyngeal squamous cell carcinomas (SCCs) and robust prognostic biomarkers in risk-stratifying in individuals with these malignancies (5), play only a marginal role in OCSCC (6). Thus, since not all OCSCCs are attributable to tobacco and alcohol exposure, the aetiopathogenesis of these neoplasms remains unknown in several cases and no reliable biomarker capable of stratifying the prognosis of OCSCC exists. It is, therefore, of paramount importance to identify biomarkers and molecular signatures predicting cancer relapse that may guide surveillance follow-up strategies and adjuvant treatments.

The infinite proliferation of malignant cells is a hallmark of oncogenesis and telomere/telomerase interplay dictates cell replicative capacity. Telomerase is indeed usually repressed in normal somatic cells, but it is detectable in the vast majority of tumours (7, 8). By synthesizing the telomere sequences and thus preventing cell senescence and apoptosis, the inappropriate activation of the catalytic component of the telomerase, telomerase reverse transcriptase (TERT), appears crucial for maintaining cellular replicative capacity and allowing tumour formation (9). Furthermore, through its non-canonical extra-telomeric functions, the re-activation of telomerase in cancer cells may affect cancer progression and metastasis (10, 11). These properties make TERT a potentially attractive biomarker in cancer.

Among the different mechanisms leading to the inappropriate reactivation of TERT in cancer, mutually exclusive recurrent C-to-T transitions at nucleotides 1,295,228 (-124 C>T) and 1,295,250 (-146 C>T) within the core promoter of TERT creating de novo binding sites for E-twenty-six (ETS) transcription factors and leading to increased TERT gene expression are particularly interesting: first, their prognostic role was consistently observed in several cancers (12), second, among SCCs of the UADT, TERT promoter mutations were observed to be topographically restricted to OCSCC (13), and third, unlike assessing TERT mRNA levels, TERT promoter mutations can be more easily analysed in formalin-fixed paraffin-embedded (FFPE) specimens from routinely collected biopsies.

Although a previous investigation conducted in a population of subject with OCSCC from Taiwan found that those harbouring the -124 C>T TERT promoter mutation had a worse prognosis, this was not statistically significant. However, current evidence suggests that the effect of TERT promoter mutations may be affected by a common single nucleotide polymorphism (SNP), rs2853669, within the TERT core promoter close to the hotspot mutation sites (14). The minor C-variant allele of the SNP disrupts a pre-existing ETS2 binding site at -245 bp in the TERT promoter region resulting in decreased TERT expression (15) and thus, counteracts the transactivation activity of the TERT promoter hotspots (14). A meta-analysis reports that among cancer patients with TERT promoter mutations, the rs2853669 T/T genotype confers a worse prognosis (16), but the modifying role of this SNP in the prognostic value of TERT promoter mutations is still controversial (12, 17–20). To date, the prognostic value of rs2853669 in OCSCC remains to be elucidated.

Thus, the main aims of this study were to investigate the prevalence and the clinical significance of TERT promoter mutations and the impact of the TERT rs2853669 SNP in a larger series of patients surgically treated for OCSCC.

In the present investigation, we observed that approximately one-third of OCSCC samples harboured TERT promoter mutations with the -124 C>T mutation having a significant adverse impact on the outcome; particularly, when coexisting with the T/T genotype of rs2853669, -124 C>T mutation increased the risk of death by 4 times.

In the literature, the frequency of TERT promoter mutations in OCSCC varies significantly among studies ranging from 30.4 to 75% (13, 28–34). This variability could be attributable to different patient population characteristics or methodological approaches. In our cohort, we found 31.3% (45 of 144) of OCSCC samples harboured TERT promoter mutations, which was in line with other studies (13, 28, 30, 33, 34). In agreement with other studies on OCSCC (20, 28–32), the two mutations have different frequency, with a higher prevalence of -124 C>T (29 of 144) compared to -146 C>T (16 of 144).

With respect to oncological outcomes, an important finding emerging from this study is that the two somatic TERT promoter mutations displayed different behaviour. Indeed, while patients with the -124 C>T TERT promoter mutation had a higher risk of mucosal failure and poorer DFS and OS, patients with tumours harbouring the -146 C>T mutation had an improved clinical outcome, similar to those with unmutated TERT promoter. The recruitment of the transcription factor GABPA, a member of ETS family, specifically to mutant TERT promoters mediates long-range chromatin interaction and enrichment of active histone marks, and hence drives TERT transcription (35). Although both the -124 C>T and -146 C>T mutations generate identical sequences, enable binding of GABPA transcription factors, and are equally efficient in increasing TERT transcription in vitro (36), previous reports demonstrated that these mutations are not functionally identical. Indeed, a peculiar pathway of activation by non-canonical NF-ĸB signalling was only described for the -146 C>T mutation (37, 38). In addition, in vivo, the -124 C>T mutation was associated with higher TERT expression/telomerase activity compared to -146 C>T (39, 40). A significant body of evidence has demonstrated that high levels of tumour TERT expression and/or telomerase activity are significantly associated with aggressiveness of disease, advanced clinical stage, and poor OS and/or DFS in several types of tumours, including UADT SCC (13, 26, 27, 41). The mechanism(s) by which high TERT expression ultimately facilitates cancer progression and constitutes a prognostic factor are not completely elucidated, and seems not be attributable only to TERT’s ability to maintain telomere length. Indeed, accumulating evidence suggests that TERT may also contribute to carcinogenesis via telomere length-independent mechanisms, including enhancement of proliferation, resistance to apoptosis, inflammation, invasion and metastasis altogether contributing towards a more aggressive phenotype of cancer cells (10, 11, 42–50). Therefore, it is conceivable that the -124 C>T TERT promoter mutation, inducing higher expression of TERT in the tumour, results in the increased severity of disease as we observed in our cohort of OCSCC patients. Corroborating our results, Arantes et al. (33) found that the -124 C>T TERT promoter mutation was associated with increased risk of tumour relapse and death in a cohort of 88 Brazilian patients with SCC of the UADT. However, other studies in different tumour types have reported contradicting clinical effects of TERT promoter mutations, ranging from poorer survival associated with the -146 C>T TERT promoter mutation to unchanged clinical outcome (28, 29, 32, 51–54). Given that the two mutations create an identical sequence corresponding to a de novo binding site for ETS transcription factors, these alternative results may depend on the genetic context, including the SNP background in which TERT mutations arise.

For the common polymorphism rs2853669 T>C, which disrupts a pre-existing ETS2 binding site within the TERT core promoter, controversial clinical impacts have been reported (12, 17–20). Our study demonstrates for the first time that the rs2853669 T/T genotype influences the clinical outcome of OCSCC patients, being significantly associated with increased risk of disease progression. Importantly, the coexistence of the T/T genotype of rs2853669 and the -124 C>T TERT promoter mutation is associated with a significantly poorer prognosis including mucosal failure, disease progression and death. The effect of the rs2853669 SNP may be related to higher telomerase activity and TERT expression conferred by the T/T genotype (15) that can also additionally intensify the transactivation activity of TERT promoter mutations (14). Thus, we can speculate that high TERT levels conferred by the -124 C>T TERT promoter mutation and/or rs2853669 T/T genotype may promote tumour progression, probably as a consequence of the extra-telomeric non-canonical functions of telomerase. Unfortunately, we did not have enough tumour material to contemporaneously analyse TERT promoter status and TERT expression/activity, and further studies should be undertaken to extend and validate these findings.

A secondary finding of our study was the absent of TERT promoter mutations in the matched adjacent mucosa. This partly differs from a previous study by Chang et al. (29), and may be due to the reduced number of adjacent normal mucosal specimens available in our cohort. Nonetheless, the finding that metastatic and recurrent head and neck squamous cell carcinomas have more TERT promoter mutations compared to primary tumours (31) suggests that the acquisition of these mutations is a late event in carcinogenesis, and may explain the lack of TERT promoter mutations in the tumour’s adjacent mucosa.

Interestingly, we confirmed that telomeres in mucosa adjacent to TERT promoter mutated tumours were significantly shorter than those adjacent to tumours retaining unmutated TERT promoter (13), and additionally we found that the mucosa adjacent to -124 C>T mutated tumours had the shortest telomeres. As critically short telomeres are a hallmark of genomic instability associated to carcinogenesis and may be considered a marker of field cancerization (26, 27), is not surprising that patients harbouring the -124 C>T TERT promoter mutation showed a significantly increased risk of tumour relapse. These data, for the first time, support a prognostic role for tumour relapse of the -124 C>T TERT promoter mutation in patients with OCSCC likely related to the very short telomeres in the mucosa surrounding the tumour in which the mutation arises.

In conclusion, we found that the -124 C>T TERT promoter mutation, as well as the T/T genotype of the rs2853669 SNP, may be a risk factor for the aggressiveness of OCSCC, and the coexistence of these genetic variations might represent a greater risk of adverse outcome. Supported by the fact that the clinical significance of this mutation is consistent with the biological properties of TERT, that TERT promoter mutations were found to stratify the prognosis in several other cancers, are easy to identify using tissue from routinely collected biopsies and address the unmet clinical need of having a validated prognostic marker for OCSCC, our observations raise the possibility that the -124 C>T TERT promoter mutation in combination with the SNP rs2853669 T/T genotype may serve as a valuable prognostic marker in this cancer, with the ability to guide therapeutic and follow-up strategies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Treviso/Belluno provinces (protocol code 346/AULSS9, March 10, 2015), Italy. The patients/participants provided their written informed consent to participate in this study.

Conceptualization: ADR, PBR. and SG. Methodology: SG. Statistical analysis: JP. Investigation: SG, PBR, ER, GT, LA, RDC, MR, PN, AM, VC, MT, LB, GS, EE, MMa, MS, RB, APDT, MG, MMo, and GE. Resources: ADR. Writing-original draft preparation: SG and PBR. Writing-review and editing: SG, PBR, JF, and ADR. Supervision: ADR. Project administration: PBR and SG. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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