Telomerase reverse transcriptase (TERT) promoter mutations (TPMs) are frequently found in different cancer types, including ∼70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma-sequencing datasets, where melanomas with BRAF V600E mutations were more likely to have a CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

Telomerase activity and telomere elongation are essential conditions for the unlimited proliferation of neoplastic cells. Point mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been found to occur at high frequencies in several tumour types and considered a primary cause of telomerase reactivation in cancer cells. These mutations promote TERT gene expression by multiple mechanisms, including the generation of novel binding sites for nuclear transcription factors, displacement of negative regulators from DNA G-quadruplexes, recruitment of epigenetic activators and disruption of long-range interactions between TERT locus and telomeres. Furthermore, TERT promoter mutations cooperate with TPP1 promoter nucleotide changes to lengthen telomeres and with mutated BRAF and FGFR3 oncoproteins to enhance oncogenic signalling in cancer cells. TERT promoter mutations have been recognized as an early marker of tumour development or a major indicator of poor outcome and reduced patients survival in several cancer types. In this review, we summarize recent findings on the role of TERT promoter mutations, telomerase expression and telomeres elongation in cancer development, their clinical significance and therapeutic opportunities.

BACKGROUND: Tumor genotyping is becoming crucial to optimize the clinical management of patients with advanced differentiated thyroid cancer (DTC); however, its implementation in clinical practice remains undefined. We herein report our single-center experience on molecular advanced DTC testing by next-generation sequencing approach, to better define how and when tumor genotyping can assist clinical decision making.
METHODS: We retrospectively collected data on all adult patients with advanced DTC who received molecular profiling at the IRCSS Sant\'Orsola-Malpighi Hospital from 2008 to 2022. The genetic alterations were correlated with radioactive iodide refractory (RAI-R), RAI uptake/disease status, and time to RAI resistance (TTRR) development.
RESULTS: A significant correlation was found between RAI-R development and genetic alterations (P = 0.0001). About 48.7% of RAI-R cases were positive for TERT/TP53 mutations (as both a single event and comutations with other driver gene alterations, such as BRAF mutations, RAS mutations, or gene fusions), while the great majority of RAI-sensitive cases carried gene fusions (41.9%) or were wild type (WT; 41.9%). RAI uptake/disease status and time to TTRR were significantly associated with genetic alterations (P = 0.0001). In particular, DTC with TERT/TP53 mutations as a single event or as comutations displayed a shorter median TTRR of 35.4 months (range 15.0-55.8 months), in comparison to the other molecular subgroups. TERT/TP53 mutations as a single event or as comutations remained independently associated with RAI-R after Cox multivariate analysis (hazard ratio 4.14, 95% CI 1.51-11.32; P = 0.006).
CONCLUSIONS: Routine testing for genetic alterations should be included as part of the clinical workup, for identifying both the subset of more aggressive tumors and the subset of tumors harboring actionable gene fusions, thus ensuring the appropriate management for all patients with advanced DTC.

The American Thyroid Association risk stratification (ATA) and the American Joint Committee on Cancer Tumor Node Metastases (TNM) predict recurrence and mortality of differentiated thyroid cancer (DTC). BRAFV600E and TERT promoter mutations have been shown to correlate with the histopathological features and outcome of DTC. Our objectives were to study the correlation of these molecular markers with these clinicopathological-staging systems.
We studied 296 unselected patients, 214 females and 82 males with a median age of 36 years (IQR 23.3-49.0). BRAFV600E and TERT promoter mutations were tested by PCR-based Sanger sequencing. Data were extracted from medical records and analysed using Chi-Square and Fisher Exact tests and Kaplan Meier analysis.
Of 296 patients tested, 137 (46.3%) had BRAFV600E-positive tumors and 72 (24.3%) were positive for TERT promoter mutations. The BRAFV600E mutation did not correlate with the ATA and TNM staging, being non-significantly different in various stages of these systems and did not predict the development of persistent disease (PD) (P 0.12). Unlike BRAFV600E, TERT promoter mutations were more frequent in the ATA high-risk than in intermediate- or low-risk tumors (P 0.006) and in TNM stages III and IV than lower stages (P <0.0001). TERT promoter mutations also predicted the outcome, being present in 37.2% of patients with PD compared to only 15.4% in those without evidence of disease (P <0.0001). The same pattern was also seen when BRAFV600E and TERT promoter mutations were combined.
TERT promoter mutations alone or in combination with BRAFV600E mutation, but not BRAFV600E mutation alone, correlated well with the ATA and TNM staging and predicted development of PD, especially in higher stages of these systems.

Melanoma is the most aggressive form of skin cancer and the leading cause of death from cutaneous tumors. Several studies have associated alterations in the TERT promoter region (pTERT) with gene overexpression, aggressiveness and poor prognosis of the disease. The aim of this study was to clarify the role of pTERT molecular status in paired samples of primary melanoma and metastasis using tissue and plasma to establish a correlation with disease progression and survival. A total of 88 FFPE tissue samples from 53 patients with advanced melanoma were analyzed. Of these, 35 had paired samples. We also examined cfDNA samples from plasma of 25 patients. We detected a good correlation between primary tumors and metastases in pTERT mutation and methylation status. We were also able to identify pTERT mutations in plasma samples that correlated with mutational status in tissue samples. Interestingly, the C250T mutation was associated with worse survival and higher TERT mRNA expression, compared to the other most common mutation: C228T. In addition, hyper-methylation of the promoter region seems to be related to the progression of pTERT wild type (WT) patients. These results suggest that TERT gene alterations plays an important role during tumor progression, with the detection of the C250T mutation in tissue and plasma as a potential biomarker of poor prognosis in patients with advanced melanoma.

Thyroid cancers are often initiated by the acquisition of a BRAFV600E mutation. BRAFV600E-driven thyroid tumors display a wide range of behaviors, from the slow-growing papillary carcinomas to the highly aggressive anaplastic. Mutations in the promoter of TERT (telomerase reverse transcriptase) gene were discovered a decade ago and identified as prevalent events in thyroid cancers. Multiple studies showed that TERT promoter mutations, particularly when co-occurring with BRAFV600E, are markers of poor prognosis across thyroid cancer subtypes, and can be implemented for routine clinical stratification. Mechanistically, TERT promoter mutations reactivate telomerase expression via the differential recruitment of transcriptional complexes. Re-expression of TERT impacts tumor biology, plausibly via both the well-known function of telomerase maintaining telomeres and by affecting other cancer-relevant processes.

The incidence of thyroid cancer in China has rapidly increased in recent decades. As the genetic profiles of thyroid cancer vary dramatically between different geographical regions, a comprehensive genetic landscape of thyroid cancer in the Chinese population is urgently needed.
We retrospectively included thyroid cancer patients from three Chinese medical centers between February 2015 and August 2020. To dissect the genomic profiling of these patients, we performed targeted next-generation sequencing on their tumor tissues using a 1,021-gene panel.
A total of 458 Chinese patients with thyroid cancer were enrolled, including four malignant histological subtypes arising from follicular epithelial thyroid cells. BRAF driver mutations were identified in 76.0% of patients, followed by RET rearrangements (7.6%) and RAS driver mutations (4.1%). Tumors with more somatic mutations correlated with worse clinical characteristics, including older age at diagnosis, less differentiation of tumor, larger tumor size, lymph node metastasis and distal metastasis. Subclonal BRAF mutations occurred in 20% (6/30) of patients and were frequent in poorly differentiated or anaplastic tumors (33.3% [2/6] vs. 4.2% [1/24], P = 0.09) and those with distal metastasis (50.0% [2/4] vs. 8.7% [2/23], P = 0.09). Tumors with TERT promoter mutations had significantly more somatic mutations (average: 6.5 vs. 1.8, P < 0.001). Moreover, TERT promoter mutations were not associated with lymph node metastasis but significantly associated with older age at diagnosis and poorly differentiated or anaplastic tumors, regardless of their clonal architecture.
Our results shed light on the molecular pathogenesis and clinical characteristics of thyroid cancer in the Chinese population. The number of somatic mutations, TERT promoter mutations, and the clonal architecture of BRAF mutations should be considered in the risk stratification of thyroid cancer.

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter region are highly frequent in urothelial cancer (UC), and their detection in urine (cell-free DNA from the urine supernatant or DNA from exfoliated cells in the urine pellet) has demonstrated promising evidence as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumour-derived mutations in urine requires highly sensitive methods, capable of measuring low-allelic fraction mutations. We developed sensitive droplet digital PCR (ddPCR) assays for detecting urinary TERT promoter mutations (uTERTpm), targeting the two most common mutations (C228T and C250T), as well as the rare A161C, C228A, and CC242-243TT mutations. Here, we described the step-by-step protocol uTERTpm mutation screening using simplex ddPCR assays and give some recommendations for isolation of DNA from urine samples. We also provide limits of detection for the two most frequent mutations and discuss advantages of the method for clinical implementation of the assays for the detection and monitoring of UC.

Circulating tumor cells (CTCs) are one of the most important causes of tumor recurrence and distant metastases. Glioblastoma (GBM) has been considered restricted to the brain for many years. Nevertheless, in the past years, several pieces of evidence indicate that hematogenous dissemination is a reality, and this is also in the caseof GBM. Our aim was to optimize CTCs\' detection in GBM and define the genetic background of single CTCs compared to the primary GBM tumor and its recurrence to demonstrate that CTCs are indeed derived from the parental tumor. We collected blood samples from a recurrent IDH wt GBM patient. We genotyped the parental recurrent tumor tissue and the respective primary GBM tissue. CTCs were analyzed using the DEPArray system. CTCs Copy Number Alterations (CNAs) and sequencing analyses were performed to compare CTCs\' genetic background with the same patient\'s primary and recurrent GBM tissues. We identified 210 common mutations in the primary and recurrent tumors. Among these, three somatic high-frequency mutations (in PRKCB, TBX1, and COG5 genes) were selected to investigate their presence in CTCs. Almost all sorted CTCs (9/13) had at least one of the mutations tested. The presence of TERT promoter mutations was also investigated and C228T variation was found in parental tumors and CTCs (C228T heterozygous and homozygous, respectively). We were able to isolate and genotype CTCs from a patient with GBM. We found common mutations but also exclusive molecular characteristics.

To estimate the prevalence of two most common and mutually exclusive -124 C > T and -146 C > T TERT promoter mutations in HNSCC and analyse their prognostic role.
The databases Medline (via Ovid), Embase (via Ovid), Cochrane Library, Scopus, and Web of Science (Core Collection) were searched from inception to December 2022 to identify studies analysing TERT promoter mutations in HNSCC. Pooled prevalence of TERT promoter mutations and hazard ratio (sHR) of death/progression, with corresponding confidence intervals (CI), were estimated.
The initial search returned 6416 articles, of which 17 studies, including 1830 patients, met the criteria for prevalence meta-analysis. Among them, 8 studies fitted the inclusion criterion to analyse the prognostic impact of TERT promoter mutations. Overall, 21% (95% CI: 12%-31%) of HNSCCs harboured TERT promoter mutation. TERT promoter mutations were more commonly found in oral cavity cancer (prevalence = 47%, 95% CI: 33%-61%), followed by laryngeal/hypopharyngeal cancer (prevalence = 12%, 95% CI: 4%-25%), while they were quite rare in oropharyngeal cancer (prevalence = 1%, 95% CI: 0%-4%). TERT promoter mutation -124 C > T was associated with a higher risk of death (sHR = 2.01, 95% CI: 1.25-3.23) and progression (sHR = 2.79, 95% CI: 1.77-4.40), while -146 C > T TERT promoter mutation did not show any significant correlation neither to overall nor progression-free survival.
TERT promoter mutations were mainly topographically restricted to oral cavity cancer. -124 C > T was the most common TERT promoter mutation and was significantly associated to worse outcome in HNSCC.