Telomerase is an enzyme made up of a catalytic subunit (telomerase reverse transcriptase or TERT) and a ribonucleic acid (TERC).1 It is known to play a major role in telomere maintenance and cell senescence. Under normal conditions, it is only expressed in germline cells, fetal tissue, and stem cells. The somatic cells of other tissues lack telomerase activity, resulting in the progressive shortening of their telomeres after repeated cell division. Many studies have shown that telomerase activity is detected in more than 90% of malignant tumors,2 including different types of skin cancer.3 In melanocyte tumors in particular, several studies have identified increased telomerase activity in the neoplastic cells, from nevi to melanomas, with the highest telomerase activity detected in metastatic melanomas.4 Furthermore, the role of TERT promoter mutations in the pathogenesis of melanoma has recently been demonstrated.5,6 Several studies have identified recurring mutations in the TERT promoter region (c.1-146C>T, c.1-124C>T, c.1-124/-125CC>TT, c.1-138/-139CC>TT) in between 22% and 71% of sporadic melanomas associated with older patients, with melanomas in areas exposed to sunlight, of the nodular histologic subtype, a higher Breslow thickness, presence of ulceration, and high mitotic index.7–9 Studies in tissue and cell lines have shown that these mutations produce increased expression of telomerase by creating new binding sites for transcription factors of the ETS family.9–12 The presence of these mutations has been shown to be an independent prognostic factor linked to reduced disease-free and overall survival.8,13 Furthermore, this link to survival has been found to vary depending on the type of mutation identified, and is modified by the presence of rs2853669 polymorphism.14,15 Recent studies have also shown the prognostic value of telomere length by identifying lower survival rates in patients with shorter telomeres.16,17

It therefore appears that telomerase expression plays a major role in melanoma carcinogenesis; however, few studies have analyzed telomerase expression at the protein level, and those studies include small series with few melanomas. Moreover, the study of telomerase expression may be of utility for stratifying patients based on prognosis. The objectives of this study are to analyze telomerase expression by means of immune staining in a series of melanomas (primary and metastatic) and melanocytic nevi, and to correlate expression levels with the clinical and pathologic characteristics and the course of the patients, and with mutations in the TERT promoter.

The results of this study show that telomerase expression occurs in all the melanocytic tumors analyzed, and that a gradually increasing trend from nevus to melanoma exists, which may be implicated in the tumor progression in this type of cancer.

In the primary melanomas in our series, we showed that the heterogeneous expression pattern is associated with more aggressive melanomas (faster-growing melanomas with a tumor thickness of more than 4mm, with mitosis, and with mutations in the TERT promoter); this has not been previously reported. These results contrast with those recently reported by Hugdahl et al,22 who describe homogeneous expression in most of their cases. Our findings, however, may support those reported by Kohli et al,23 who report heterogeneous expression of non-nuclear telomerase in melanomas but not in benign lesions. This heterogeneous expression pattern may be the result of intratumoral heterogeneity in melanoma, which has been linked to different tumor subpopulations, and is linked to resistance to systemic treatments.24,25 Furthermore, we cannot rule out the possibility that the heterogeneity observed in our study may be due to a defect in the staining procedure, although this is unlikely, as this pattern was not observed in any of the benign melanocytic lesions.

Unlike other studies, in our series, the intensity and extent of telomerase expression were independent of the clinical and pathologic characteristics. Hugdahl et al22 reported a statistically significant link between telomerase expression (44% of primary melanomas in that series) and tumor thickness, and Zygouris et al26 described a link with Breslow thickness and ulceration. Our findings contrast with several studies that have analyzed telomerase activity, such as the study by Ramírez et al, which found greater telomerase activity in melanomas with greater tumor thickness, a higher Clark score, and mitosis27; the results of Miracco et al,28 in which telomerase activity was also associated with Breslow thickness and the Clark score; and the study by Carvalho et al,29 which found greater telomerase activity in ulcerated melanomas, with a greater Breslow thickness, mitosis, vascular invasion, and satellitosis. In our series, melanomas of lentigo maligna melanoma showed greater expression intensity, although this did not reach statistical significance. This last finding does not support the findings of the series by Pópulo et al,8 in which telomerase expression was only associated with the surface spreading histologic subtype.

With respect to the mutations in the TERT promoter, in line with reporting by other authors,8,9 in a previous publication,30 we reported a prevalence of 33% and an association with ulcerated melanomas with greater tumor thickness, with mitosis, and of the nodular histologic subtype. As in other studies, in this series, we found no differences using immune staining in telomerase expression between mutated and nonmutated melanomas.8,22,23 This finding contrasts with several prior publications, which found greater telomerase expression in melanomas with mutations in the promoter.9–12 This discrepancy may be due to the fact that those studies analyzed telomerase expression at the messenger RNA level and not at the protein level, and other molecular mechanisms may therefore exist that regulate telomerase expression. Indeed, several studies have focused on epigenetic regulation of telomerase expression. It has been reported that post-transcriptional regulation of the TERT through microRNA expression may suppress telomerase expression31,32 and that hypermethylation in the DNA of the TERT gene may increase telomerase expression.33 It may therefore be useful to measure the length of the telomeres and/or telomerase activity and correlate it to the presence of the mutation in order to determine whether only mutated genes show increased activity or whether wild-type genes also show greater telomerase activity through these other molecular mechanisms.

Furthermore, we have identified variable levels of telomerase expression in a series of 22 melanocytic nevi with mild expression in most of the samples, and a tendency for expression to increase from intradermal nevi to dysplastic nevi. It should also be noted that, in all the melanomas with a nevus precursor, telomerase expression in the accompanying nevus was much less intense than in the tumor cells of the melanoma. These results suggest a potential role of telomerase in tumor progression in melanocytic cancers. Several authors have analyzed telomerase activity in different types of melanocytic tumor and have found that telomerase expression increases progressively from common nevi to dysplastic nevi and melanomas.4,34 These differences in telomerase expression between nevus and melanoma, however, have been found not to be of use in the differential diagnosis, as expression may be variable in both types of tumor.30

In this study, telomerase expression was analyzed using immune staining, unlike most previously published studies, which analyzed telomerase activity using the telomere repeat amplification protocol (TRAP).4,23–25 Immune staining has the advantage of being a simple and cheap method that is performed using paraffinized samples and makes it possible to visualize the intensity and extent of expression, not only in the cells of interest but also in adjacent healthy skin. Indeed, in line with what has already been described,30 we have identified variable levels of telomerase expression, not only in the tumor cells but also in the epidermis, hair follicles, glands, dermal blood vessels, and even in the accompanying inflammatory infiltrate.

In conclusion, the results of this study show that telomerase expression occurs in all the analyzed melanocytic tumors, with greater expression in the melanomas than in the nevi. In the case of the primary melanomas, heterogeneous expression is linked to a more aggressive phenotype; however, expression intensity is independent of the clinical and pathologic characteristics of the tumors. It has also been shown that no differences exist in telomerase expression based on the mutational state of the TERT gene and other molecular mechanisms must therefore exist that regulate expression of the TERT gene in the melanomas.

This study was carried out with grants awarded by Fundación Piel Sana (EUROMELANOMA 2016 grant from the AEDV Foundation), Carlos III Institute of Health (ISCII) (PI16/01559), IISLaFe (2014/0370), and the Department of Education, Research, Culture and Sport of the autonomous government of Valencia (GV/2016/064).

The authors declare that they have no conflicts of interest.