Wolf in sheep's clothing - Oral proliferative verrucous leukoplakia: Progression with longitudinal molecular insights.

BACKGROUND: Oral proliferative verrucous leukoplakia (OPVL) is a chronic form of oral leukoplakia that progresses to a multifocal disease with confluent, exophytic and proliferative features. The clinical differential diagnosis for OPVL includes frictional keratosis, leukoplakia, chronic hyperplastic candidiasis, squamous papilloma, verrucous hyperplasia, verrucous carcinoma and squamous cell carcinoma. In this study, we aimed to delineate the dynamic changes in molecular signature during OPVL progression. We compare to a cohort of oral cavity keratinizing squamous cell carcinoma (OSCC) patients covering the spectrum of verrucous carcinoma to invasive squamous cell carcinoma including cytologically bland cuniculatum variant. METHODS: Samples from a large OPVL lesion that exhibited a histopathologic continuum of OPVL progression. RESULTS: Canonical hotspot TERT promoter mutations were identified in all patients. TERT C228T was dominant and mutually exclusive with TERT C250T. In patients with TERT C250T, there was concurrent PI3 point mutation. TP53 mutations were also consistently found (8/10). At the protein level, p53 was abnormal, with loss of function and gain of function. CONCLUSIONS: OPVL is a pathology that shows proximity to the gene expression profile of OSCC, highlighting signatures in common that can be important targets for drug treatment, as well as in the development of diagnostic and prognostic strategies for this disease.

Innovative Approach to Isolate and Characterize Glioblastoma Circulating Tumor Cells and Correlation with Tumor Mutational Status.

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.

TERT Promoter Mutations as Simple and Non-Invasive Urinary Biomarkers for the Detection of Urothelial Bladder Cancer in a High-Risk Region.

Bladder cancer (BC) is the 10th most common cancer in the world. While there are FDA-approved urinary assays to detect BC, none have demonstrated sufficient sensitivity and specificity to be integrated into clinical practice. Telomerase Reverse Transcriptase (TERT) gene mutations have been identified as the most common BC mutations that could potentially be used as non-invasive urinary biomarkers to detect BC. This study aims to evaluate the validity of these tests to detect BC in the Kerman province of Iran, where BC is the most common cancer in men. Urine samples of 31 patients with primary (n = 11) or recurrent (n = 20) bladder tumor and 50 controls were prospectively collected. Total urinary DNA was screened for the TERT promoter mutations (uTERTpm) by Droplet Digital PCR (ddPCR) assays. The performance characteristics of uTERTpm and the influence by disease stage and grade were compared to urine cytology results. The uTERTpm was 100% sensitive and 88% specific to detect primary BC, while it was 50% sensitive and 88% specific in detecting recurrent BC. The overall sensitivity and specificity of uTERTpm to detect bladder cancer were 67.7% and 88.0%, respectively, which were consistent across different tumor stages and grades. The most frequent uTERTpm mutations among BC cases were C228T (18/31), C250T (4/31), and C158A (1/31) with mutant allelic frequency (MAF) ranging from 0.2% to 63.3%. Urine cytology demonstrated a similar sensitivity (67.7%), but lower specificity (62.0%) than uTERTpm in detecting BC. Combined uTERTpm and urine cytology increased the sensitivity to 83.8%, but decreased the specificity to 52.0%. Our study demonstrated promising diagnostic accuracy for the uTERTpm as a non-invasive urinary biomarker to detect, in particular, primary BC in this population.

Occurrence, functionality and abundance of the TERT promoter mutations.

Telomere shortening at chromosomal ends due to the constraints of the DNA replication process acts as a tumor suppressor by restricting the replicative potential in primary cells. Cancers evade that limitation primarily through the reactivation of telomerase via different mechanisms. Mutations within the promoter of the telomerase reverse transcriptase (TERT) gene represent a definite mechanism for the ribonucleic enzyme regeneration predominantly in cancers that arise from tissues with low rates of self-renewal. The promoter mutations cause a moderate increase in TERT transcription and consequent telomerase upregulation to the levels sufficient to delay replicative senescence but not prevent bulk telomere shortening and genomic instability. Since the discovery, a staggering number of studies have resolved the discrete aspects, effects and clinical relevance of the TERT promoter mutations. The promoter mutations link transcription of TERT with oncogenic pathways, associate with markers of poor outcome and define patients with reduced survivals in several cancers. In this review, we discuss the occurrence and impact of the promoter mutations and highlight the mechanism of TERT activation. We further deliberate on the foundational question of the abundance of the TERT promoter mutations and a general dearth of functional mutations within noncoding sequences, as evident from pan-cancer analysis of the whole-genomes. We posit that the favorable genomic constellation within the TERT promoter may be less than a common occurrence in other noncoding functional elements. Besides, the evolutionary constraints limit the functional fraction within the human genome, hence the lack of abundant mutations outside the coding sequences.

TERT Promoter Mutation as a Potential Predictive Biomarker in BCG-Treated Bladder Cancer Patients.

Telomerase reverse transcriptase gene promoter (TERTp) mutations are recognized as one of the most frequent genetic events in bladder cancer (BC). No studies have focused on the relevance of TERTp mutations in the specific group of tumors treated with Bacillus Calmette-Guérin (BCG) intravesical therapy. Methods - 125 non muscle invasive BC treated with BCG therapy (BCG-NMIBC) were screened for TERTp mutations, TERT rs2853669 single nucleotide polymorphism, and Fibroblast Growth Factor Receptor 3 (FGFR3) hotspot mutations. Results - TERTp mutations were found in 56.0% of BCG-NMIBC and were not associated with tumor stage or grade. FGFR3 mutations were found in 44.9% of the cases and were not associated with tumor stage or grade nor with TERTp mutations. The TERT rs2853669 single nucleotide polymorphism was associated with tumors of higher grade. The specific c.1-146G>A TERTp mutation was an independent predictor of nonrecurrence after BCG therapy (hazard ratio-0.382; 95% confidence interval-0.150-0.971, p = 0.048). Conclusions - TERTp mutations are frequent in BCG-NMIBC and -146G>A appears to be an independent predictive marker of response to BCG treatment with an impact in recurrence-free survival.

Combined genetic and epigenetic alterations of the TERT promoter affect clinical and biological behavior of bladder cancer.

In urothelial bladder cancer (UBC), risk stratification remains an important unmet need. Limitless self-renewal, governed by TERT expression and telomerase activation, is crucial for cancer progression. Thus, telomerase activation through the interplay of mutations (TERTpMut ) and epigenetic alterations in the TERT promoter may provide further insight into UBC behavior. Here, we investigated the combined effect of TERTpMut and the TERT Hypermethylated Oncological Region (THOR) status on telomerase activation and patient outcome in a UBC international cohort (n = 237). We verified that TERTpMut were frequent (76.8%) and present in all stages and grades of UBC. Hypermethylation of THOR was associated with higher TERT expression and higher-risk disease in nonmuscle invasive bladder cancers (NMIBC). TERTpMut alone predicted disease recurrence (HR: 3.18, 95%CI 1.84 to 5.51, p < 0.0001) but not progression in NMIBC. Combined THORhigh /TERTpMut increased the risk of disease recurrence (HR 5.12, p < 0.0001) and progression (HR 3.92, p = 0.025). Increased THOR hypermethylation doubled the risk of stage progression of both TERTpwt and TERTpMut NMIBC. These results highlight that both mechanisms are common and coexist in bladder cancer and while TERTpMut is an early event in bladder carcinogenesis THOR hypermethylation is a dynamic process that contributes to disease progression. While the absence of alterations comprises an extremely indolent phenotype, the combined genetic and epigenetic alterations of TERT bring additional prognostic value in NMIBC and provide a novel insight into telomere biology in cancer.

Activation of mutant TERT promoter by RAS-ERK signaling is a key step in malignant progression of BRAF-mutant human melanomas.

Although activating BRAF/NRAS mutations are frequently seen in melanomas, they are not sufficient to drive malignant transformation and require additional events. Frequent co-occurrence of mutations in the promoter for telomerase reverse transcriptase (TERT), along with BRAF alterations, has recently been noted and correlated with poorer prognosis, implicating a functional link between BRAF signaling and telomerase reactivation in melanomas. Here, we report that RAS-ERK signaling in BRAF mutant melanomas is critical for regulating active chromatin state and recruitment of RNA polymerase II at mutant TERT promoters. Our study provides evidence that the mutant TERT promoter is a key substrate downstream of the RAS-ERK pathway. Reactivating TERT and hence reconstituting telomerase is an important step in melanoma progression from nonmalignant nevi with BRAF mutations. Hence, combined targeting of RAS-ERK and TERT promoter remodeling is a promising avenue to limit long-term survival of a majority of melanomas that harbor these two mutations.

[BRAF V600E Mutation and TERT Promoter Mutation in Papillary Thyroid Carcinomas and Their Association with Clinicopathological Characteristics].

OBJECTIVE: To explore the relationships of BRAF V600E and TERT promoter mutations with the clinicopathological features in papillary thyroid carcinoma (PTC). METHODS: The mutations of BRAF V600E and TERT promoters were examined by PCR-direct sequencing in tumor tissues from 326 PTC patients, while the relationships between the gene mutations and clinicopathological features were analyzed. RESULTS: BRAF V600E mutation was found in 269/326 (82.52%), and TERT promoter mutation in 11/326 (3.37%) of PTC patients. In site mutations of TERT promoter, 9 cases were C228T and 2 cases were C250T. Single factor analysis showed that BRAF V600E mutations were significantly associated with age and recurrence/distant metastasis of tumor (P < 0.05), while TERT promoter mutations were significantly associated with age, tumor size, extrathyroidal extension, T stage, AJCC stage and recurrence/distant metastasis of tumor (P < 0.05). Coexistence of BRAF V600E and TERT promoter mutations (BRAF+/TERT+) were particularly associated with age, tumor size, extrathyroidal extension, T stage and AJCC stage (P < 0.05). CONCLUSION: Coexistence of BRAF V600E and TERT promoter mutations in PTC shows more aggressive tumor behavior.

Telomerase reverse transcriptase promoter alterations across cancer types as detected by next-generation sequencing: A clinical and molecular analysis of 423 patients.

BACKGROUND: Telomerase reverse transcriptase (TERT) promoter mutations that may affect telomerase activity have recently been described in human malignancies. The purpose of this study was to investigate the clinical correlates of TERT promoter abnormalities in a large cohort of patients with diverse cancers. METHODS: This study analyzed TERT promoter alterations and clinical characteristics of 423 consecutive patients for whom molecular testing by next-generation sequencing was performed between August 2014 and July 2015. RESULTS: Of the 423 patients, 61 (14.4%) had TERT promoter mutations, and this placed TERT promoter alterations among the most prevalent aberrations after tumor protein 53 (TP53; 39%) and KRAS and cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) alterations (15% each) in this population. TERT promoter alterations were more frequent in men (P = .031) and were associated with brain cancers (P = .001), skin cancers/melanoma (P = .001), and a higher number of aberrations (P = .0001). A co-alteration analysis found that TERT promoter alterations were significantly correlated with CDKN2A/B (P = .001) and BRAF abnormalities (P = .0003). Patients harboring TERT promoter alterations or TP53 or CDKN2A/B alterations and those with 4 or more alterations demonstrated shorter survival (hazard ratio for normal TERT promoters vs aberrant ones, 0.44; P = .017). However, only a higher number of alterations remained significant in the multivariate analysis. CONCLUSIONS: Overall, TERT promoter alterations were among the most prevalent aberrations in this population, with very high rates in brain cancers (48% of patients) and melanomas (56% of patients). These aberrations frequently coexist with a high number of other aberrations, with the latter feature also significantly associated with poorer overall survival. Therapeutic options for targeting tumors with TERT promoter mutations are currently limited, although a variety of novel approaches are under development. Cancer 2018;124:1288-96. © 2017 American Cancer Society.

Telomerase and N-Cadherin Differential Importance in Adrenocortical Cancers and Adenomas.

Adrenocortical carcinomas (ACC) are most frequently highly aggressive tumors. We assessed the telomerase reverse transcriptase (TERT) and N-cadherin role in the biology of ACC and their potential utility as molecular biomarkers, in different types of tumoral adrenocortical tissue. A total of 48 adrenal cortex samples (39 tumoral and 9 normal adrenal glands) were studied. TERT promoter mutations were searched by PCR and Sanger sequencing in two hotspots positions (-124 and -146). Also, telomerase and N-cadherin expression were evaluated by immunohistochemistry. TERT promoter mutations were not detected in any of the samples either malignant or benign. Telomerase nuclear expression was present in 26.6% of ACC and in 45.5% of non-functioning adenomas. It was absent in benign Cushing's lesions and in normal adrenal glands. Contrarily, N-cadherin was always expressed in the cellular membranes of benign adenomas or normal adrenals but no expression was detected in the majority of ACC. Nuclear telomerase and membrane N-cadherin expression were positively correlated in ACCs. We conclude that in ACC, the loss of N-cadherin is a frequent phenomenon while the existence of TERT promoter mutations is not and nuclear telomerase expression is present in only a minority of cases. Since the loss of N-cadherin expression was identified in both high and low proliferative ACC, this marker should be considered important for diagnostic application. Our study also suggests the existence of a TERT non-canonical function in cell adhesion. J. Cell. Biochem. 118: 2064-2071, 2017. © 2017 Wiley Periodicals, Inc.

Altered TERT promoter and other genomic regulatory elements: occurrence and impact.

Study of genetic alterations, inherited or acquired, that increase the risk or drive cancers and many other diseases had remained mostly confined to coding sequences of the human genome. Data from genome wide associations studies, development of the Encyclopedia of DNA Elements (ENCODE), and a spurt in detection of driver somatic mutations have shifted focus towards noncoding regions of the human genome. The majority of genetic variants robustly associated with cancers and other syndromes identified through genome wide studies are located within noncoding regulatory regions of the genome. Genome wide techniques have put an emphasis on the role of three-dimensional chromosomal structures and cis-acting elements in regulations of different genes. The variants within noncoding genomic regions can potentially alter a number of regulatory elements including promoters, enhancers, insulators, noncoding long RNAs and others that affect cancers and various diseases through altered expression of critical genes. With effect of genetic alterations within regulatory elements dependent on other partner molecules like transcription factors and histone marks, an understanding of such modifications can potentially identify extended therapeutic targets. That concept has been augmented by the detection of driver somatic noncoding mutations within the promoter region of the telomerase reverse transcriptase (TERT) gene in different cancers. The acquired somatic noncoding mutations within different regulatory elements are now being reported in different cancers with an increased regularity. In this review we discuss the occurrence and impact of germline and somatic alterations within the TERT promoter and other genomic regulatory elements.

Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas.

OBJECTIVE: The genes causing familial nonmedullary thyroid carcinoma (FNMTC) identified to date are only involved in a small fraction of the families. Recently, somatic mutations in TERT promoter region and in EIF1AX gene were reported in thyroid tumours of undefined familial status. The aim of this study was to investigate the role of TERT and EIF1AX mutations in familial thyroid tumours. DESIGN: The promoter region of TERT was sequenced in leucocyte DNA of the probands from 75 FNMTC families. In thyroid tumours from 54 familial cases, we assessed somatic TERT promoter, RAS and BRAF hotspot mutations, and the whole EIF1AX gene. RESULTS: No potentially pathogenic germline variants were identified in TERT in the 75 FNMTC families' probands. In the 54 carcinomas, we identified five cases (9%) with hotspot somatic TERT promoter mutations. BRAF mutations were found in 41% of the tumours. All TERT-positive samples were also positive for BRAF p.Val600Glu, and this co-occurrence was found to be statistically significant (P=.008). RAS mutations were detected in four tumours wild-type for TERT (7%). Evaluation of tumour mutation data together with the patients' clinicopathological features revealed a significant correlation between TERT plus BRAF mutations and advanced tumour stage (T4) (P=.020). No mutations were identified in EIF1AX. CONCLUSIONS: The results of this study suggest that TERT promoter and EIF1AX mutations are not frequently involved in FNMTC aetiology. However, we show for the first time that TERT alterations are associated with familial thyroid tumour progression. Our data also suggest that TERT mutations are more often found in concomitance with BRAF mutations in advanced stages of FNMTC.

Molecular testing of BRAF, RAS and TERT on thyroid FNAs with indeterminate cytology improves diagnostic accuracy.

OBJECTIVE: Liquid-based (LB)-FNA is widely recognized as a reliable diagnostic method to evaluate thyroid nodules. However, up to 30% of LB-FNA remain indeterminate according to the Bethesda system. Use of molecular biomarkers has been recommended to improve its pathological accuracy but implementation of these tests in clinical practice may be difficult. Here, we evaluated feasibility and performance of molecular profiling in routine practice by testing LB-FNA for BRAF, N/HRAS and TERT mutations. METHODS: We studied a large prospective cohort of 326 cases, including 61 atypia of undetermined significance, 124 follicular neoplasms, 72 suspicious for malignancy and 69 malignant cases. Diagnosis of malignancy was confirmed by histology on paired surgical specimen. RESULTS: Mutated LB-FNAs were significantly associated with malignancy regardless of the cytological classification. Overall sensitivity was 60% and specificity 89%. Importantly, in atypia of undetermined significance and follicular neoplasm patients undergoing surgery according to the Bethesda guidelines, negative predictive values were 85.4% and 90% respectively. TERT promoter mutation was rare but very specific for malignancy (5.5%) suggesting that it could be of interest in patients with indeterminate cytology. CONCLUSIONS: Mutation profiling can be successfully performed on thyroid LB-FNA without any dedicated sample in a pathology laboratory. It is an easy way to improve diagnostic accuracy of routine LB-FNA and may help to better select patients for surgery and to avoid unnecessary thyroidectomies.

The prognostic impact of TERT promoter mutations in glioblastomas is modified by the rs2853669 single nucleotide polymorphism.

Human hotspot TERT promoter (TERTp) mutations have been reported in a wide range of tumours. Several studies have shown that TERTp mutations are associated with clinicopathological features; in some instances, TERTp mutations were considered as biomarkers of poor prognosis. The rs2853669 SNP, located in the TERT promoter region, was reported to modulate the increased TERT expression levels induced by the recurrent somatic mutations. In this study we aimed to determine the frequency and prognostic value of TERTp mutations and TERT rs2853669 SNP in 504 gliomas from Portuguese and Brazilian patients. TERTp mutations were detected in 47.8% of gliomas (216/452). Glioblastomas (GBM) exhibited the highest frequency of TERTp mutations (66.9%); in this glioma subtype, we found a significant association between TERTp mutations and poor prognosis, regardless of the population. Moreover, in a multivariate analysis, TERTp mutations were the only independent prognostic factor. Our data also showed that the poor prognosis conferred by TERTp mutations was restricted to GBM patients carrying the rs2853669 A allele and not in those carrying the G allele. In conclusion, the presence of TERTp mutations was associated with worse prognosis in GBM patients, although such association depended on the status of the rs2853669 SNP. The status of the rs2853669 SNP should be taken in consideration when assessing the prognostic value of TERTp mutations in GBM patients. TERTp mutations and the rs2853669 SNP can be used in the future as biomarkers of glioma prognosis.

TERT promoter mutations in melanoma survival.

Despite advances in targeted therapies, the treatment of advanced melanoma remains an exercise in disease management, hence a need for biomarkers for identification of at-risk primary melanoma patients. In this study, we aimed to assess the prognostic value of TERT promoter mutations in primary melanomas. Tumors from 300 patients with stage I/II melanoma were sequenced for TERT promoter and BRAF/NRAS mutations. Cumulative curves were drawn for patients with and without mutations with progression-free and melanoma-specific survival as outcomes. Cox proportional hazard regression models were used to determine the effect of the mutations on survivals. Individually, presence of TERT promoter and BRAF/NRAS mutations associated with poor disease-free and melanoma-specific survival with modification of the effect by the rs2853669 polymorphism within the TERT promoter. Hazard ratio (HR) for simultaneous occurrence of TERT promoter and BRAF/NRAS mutations for disease-free survival was 2.3 (95% CI 1.2-4.4) and for melanoma-specific survival 5.8 (95% CI 1.9-18.3). The effect of the mutations on melanoma-specific survival in noncarriers of variant allele of the polymorphism was significant (HR 4.5, 95% CI 1.4-15.2) but could not be calculated for the carriers due to low number of events. The variant allele per se showed association with increased survival (HR 0.3, 95% CI 0.1-0.9). The data in this study provide preliminary evidence that TERT promoter mutations in combination with BRAF/NRAS mutations can be used to identify patients at risk of aggressive disease and the possibility of refinement of the classification with inclusion of the rs2853669 polymorphism within TERT promoter.

Prognostic effects of TERT promoter mutations are enhanced by coexistence with BRAF or RAS mutations and strengthen the risk prediction by the ATA or TNM staging system in differentiated thyroid cancer patients.

BACKGROUND: Recent reports suggest that mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT) affect thyroid cancer outcomes. METHODS: In all, 551 patients with differentiated thyroid cancer (DTC) enrolled in this study. The median follow-up duration was 4.8 years (interquartile range, 3.4-10.6 years). RESULTS: TERT promoter mutations were detected in 25 DTCs (4.5%): 2.8% in neither BRAF-mutated nor RAS-mutated tumors, 4.8% in BRAF-mutated tumors, and 11.3% in RAS-mutated tumors. Moreover, they were frequently observed in American Thyroid Association (ATA) high-risk and TNM stage III/IV groups (9.1% and 12.9%, respectively). The coexistence of BRAF or RAS with TERT promoter mutations increased aggressive clinicopathologic features, recurrence (hazard ratio [HR] for BRAF, 4.64; 95% confidence interval [CI], 1.42-15.18; HR for RAS, 5.36; 95% CI, 1.20-24.02), and mortality (HR for BRAF, 15.13; 95% CI, 1.55-148.23; HR for RAS, 14.75; 95% CI, 1.30-167.00), even after adjustments for the age at diagnosis and sex, although the significance was lost after additional adjustments for pathologic characteristics. Furthermore, TERT promoter mutations significantly increased the risk of both recurrence and mortality in the ATA high-risk (HR for recurrence, 5.79; 95% CI, 2.07-16.18; HR for mortality, 16.16; 95% CI, 2.10-124.15) and TNM stage III/IV groups (HR for recurrence, 3.60; 95% CI, 1.19-10.85; HR for mortality, 9.06; 95% CI, 2.09-39.26). CONCLUSIONS: The coexistence of BRAF or RAS mutations enhanced the prognostic effects of TERT promoter mutations. Furthermore, TERT promoter mutations strengthened the predictions of mortality and recurrence by the ATA and TNM staging systems, particularly for high-risk patients with DTC. Cancer 2016;122:1370-1379. © 2016 American Cancer Society.

TERT promoter mutations in clear cell renal cell carcinoma.

We screened promoter region of the telomerase reverse transcriptase (TERT) for activating somatic mutations in 188 tumors from patients with clear cell renal cell carcinoma (ccRCC). Twelve tumors (6.4%) carried a mutation within the core promoter region of the gene. The mutations were less frequent in high grade tumors compared to low grade tumors [odds ratio (OR) = 0.15, 95% confidence interval (CI) = 0.03-0.72, p = 0.02]. Multivariate analysis for cause specific survival showed statistically significant poor outcome in patients with TERT promoter mutations [hazard ratio (HR) = 2.90, 95% CI = 1.13-7.39, p = 0.03]. A common polymorphism (rs2853669) within the locus seemed to act as a modifier of the effect of the mutations on patient survival as the noncarriers of the variant allele with the TERT promoter mutations showed worst survival (HR = 3.34, 95% CI = 1.24-8.98, p = 0.02). We also measured relative telomere length (RTL) in tumors and difference between tumors with and without the TERT promoter mutations was not statistically significant. Similarly, no difference in patient survival based on RTL in tumors was observed. Our study showed a relatively low frequency of TERT promoter mutations in ccRCC. Nevertheless, patients with the mutations, particularly in the absence of the rs2853669 variant showed the worst disease-specific survival. Thus, it is possible that the TERT promoter mutations define a small subset of tumors with an aggressive behavior.

Heterogeneity of TERT promoter mutations status in squamous cell carcinomas of different anatomical sites.

Squamous cell carcinoma (SCC) can arise from different anatomical sites including the skin, head and neck, lung, esophagus, genital area, and so on. Despite the same histopathologic features and immunohistochemistry profile, the SCCs of different body sites can show tremendous differences in their presenting symptoms, risk factor associations, natural history, prognosis, and response to treatment. This may reflect the fact that SCCs are heterogenous and likely have unique molecular characteristics at different anatomical sites. Recurrent somatic mutations in the TERT promoter region were first reported in human melanomas. Subsequently, other tumors including cutaneous SCC were found to demonstrate high frequencies of the same mutations. However, the incidences of TERT promoter mutation in noncutaneous SCCs have not been systemically studied. We investigated the TERT promoter mutation status among SCCs from different sites. We collected 84 cases of SCC from the skin (27), head and neck (12), lung (25), and cervix (10), as well as 10 cases of urothelial carcinoma with squamous differentiation (UC-SqD). We found that the frequencies of TERT promoter mutation among SCC of different sits are quite heterogenous: ~70% in skin SCC and UC-SqD, 16.67% in head and neck SCC, and 0% in lung and cervix SCC. These results may support the hypothesis of different carcinogenesis mechanisms of SCC in different sites. It also indicates that TERT promoter mutation could be a biomarker for distinguishing skin SCC or UC-SqD vs pulmonary SCC.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

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.