TERT Copy Research Articles

Abstract 2021: Wilms tumor and pure erythroid leukemia arising from the same clone in an adolescent

Abstract Wilms tumor and pure erythroid leukemia (PEL) are distinct malignancies arising from different tissues. Here we describe a case of an adolescent with a constitutional pathogenic BRCA2 variant who was diagnosed with a Wilms tumor and developed PEL while undergoing treatment. We provide evidence that both malignancies are distinct and arose from the same clone. A 16-year-old boy presented with a diffusely anaplastic Wilms tumor metastatic to liver. He was treated with radical nephrectomy, partial hepatectomy, radiation, and multiagent chemotherapy. Six months into treatment he developed new lung and liver lesions along with cytopenias. A bone marrow aspirate confirmed a diagnosis of PEL. His disease progressed after one cycle of myeloid leukemia-directed chemotherapy and he died of disease shortly after. Samples from his initial renal tumor, blood at Wilms tumor diagnosis, and diagnostic bone marrow for PEL were characterized by whole genome sequencing and RNA sequencing. Two sequence variants were shared in both tumors but were not found in the constitutional tissue (blood at first presentation): a promoter variant in TERT (c.-124C>T) and a missense variant in PDE4DIP (p.Gln1045His). Although neither variant has been reported in Wilms tumor or PEL, the TERT variant has been well described in other tumor types and is associated with decreased survival in some carcinomas. Both tumors also had pathogenic TP53 variants albeit a different one in each tumor (p.Arg175His in the Wilms tumor, p.Asp228* in PEL). Loss of heterozygosity at TP53 was present in both malignancies via copy number loss of 17p with a shared breakpoint. Furthermore, while both malignancies had complex and distinct karyotypes, 7p loss and 22q loss with identical breakpoints were present in both. None of these variants or copy number variants were present in constitutional tissue. RNA sequencing data confirmed the histologic finding that these were distinct malignancies as the expression patterns for each malignancy were consistent with previously published expression data for Wilms tumors and myeloid leukemias respectively. Although the patient had a constitutional pathogenic BRCA2 variant, loss of heterozygosity at that locus was not found in either tumor. Chromosome fragility testing for Fanconi anemia was uninterpretable due to the concomitant use of chemotherapeutic agents at the time of testing, however the patient had no phenotypic features to support this diagnosis. In this case there are multiple lines of evidence suggesting that a Wilms tumor and PEL arose from the same clone including shared TERT variants and copy number variant breakpoints. Kidney and hematopoietic tissue both arise from the embryonic mesoderm suggesting that the earliest alterations occurred in this tissue. The contribution of each shared pathogenic variant (TERT, BRCA2, 17p loss) to tumorigenesis and the role of chemotherapy in PEL development are still under investigation. Citation Format: Anita Villani, Winnie Lo, Yisu Li, Mohamed Abdelhaleem, Mary Shago, Federico Comitani, My Linh Thibodeau, Sarah Alexander, Uri Tabori, David Malkin, Adam Shlien, Jack Brzezinski. Wilms tumor and pure erythroid leukemia arising from the same clone in an adolescent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2021.

TERT Copy Number Alterations, Promoter Mutations and Rearrangements in Adrenocortical Carcinomas

Molecular characterization of adrenocortical carcinomas (ACC) by The Cancer Genome Atlas (TCGA) has highlighted a high prevalence of TERT alterations, which are associated with disease progression. Herein, 78 ACC were profiled using a combination of next generation sequencing (n = 76) and FISH (n = 9) to assess for TERT alterations. This data was combined with TCGA dataset (n = 91). A subset of borderline adrenocortical tumors (n = 5) and adrenocortical adenomas (n = 7) were also evaluated. The most common alteration involving the TERT gene involved gains/amplifications, seen in 22.2% (37/167) of cases. In contrast, “hotspot” promoter mutations (C > T promoter mutation at position -124, 7/167 cases, 4.2%) and promoter rearrangements (2/165, 1.2%) were rare. Recurrent co-alterations included 22q copy number losses seen in 24% (9/38) of cases. Although no significant differences were identified in cases with and without TERT alterations pertaining to age at presentation, tumor size, weight, laterality, mitotic index and Ki67 labeling, cases with TERT alterations showed worse outcomes. Metastatic behavior was seen in 70% (28/40) of cases with TERT alterations compared to 51.2% (65/127, p = 0.04) of cases that lacked these alterations. Two (of 5) borderline tumors showed amplifications and no TERT alterations were identified in 7 adenomas. In the borderline group, 0 (of 4) patients with available follow up had adverse outcomes. We found that TERT alterations in ACC predominantly involve gene amplifications, with a smaller subset harboring “hotspot” promoter mutations and rearrangements, and 70% of TERT-altered tumors are associated with metastases. Prospective studies are needed to validate the prognostic impact of these findings.

TERT and TERT promoter in melanocytic neoplasms: Current concepts in pathogenesis, diagnosis, and prognosis.

Located on chromosome locus 5p15.33, telomerase reverse transcriptase (TERT or hTERT) encodes the catalytic subunit of telomerase which permits lengthening and preservation of telomeres following mitosis. Mutations in TERT promoter (TERT-p) upregulate expression of TERT, allowing survival of malignant cells and tumor progression in wide variety of malignancies including melanoma. The objective of this review is to examine the roles of TERT and TERT-p in the pathogenesis, diagnosis, and prognostication of cutaneous melanoma. All studies of TERT or TERT-p in cutaneous melanocytic neoplasms with the following inclusion criteria were reviewed: publication date between 2010 and 2019, English language, and series of ≥3 cases were reviewed for evidence supporting the role of TERT in pathogenesis, diagnosis, and prognosis. Studies with <3 cases or focused primarily on mucosal or uveal melanocytic tumors were excluded. TERT-p mutations are frequent in chronic and non-chronic sun damage melanoma and correlate with adverse prognosis, inform pathogenesis, and may provide diagnostic support. While TERT-p mutations are uncommon in acral melanoma, TERT copy number gains and gene amplification predict reduced survival. Among atypical spitzoid neoplasms, TERT-p mutations identify biologically aggressive tumors and support the diagnosis of spitzoid melanoma. TERT-p methylation may have prognostic value in pediatric conventional melanoma and drive tumorigenesis in melanoma arising within congenital nevi. Finally, TERT-p mutations may aid in the differentiation of recurrent nevi from recurrent melanoma.

The TERT copy number gain is sensitive to telomerase inhibitors in human melanoma.

Telomerase reverse transcriptase (TERT) copy number gain is frequently observed in Asian melanoma patients. Here, we explored the correlation between TERT copy number and the effect of telomerase inhibitors in melanoma. A total of 78 melanoma cases were enrolled in the study. The TERT copy number was examined by QuantiGene Plex DNA assay. The sensitivity to telomerase inhibitors was evaluated in cell lines and patient-derived xenograft (PDX) models with or without TERT copy number gain. Among the 78 patients, 33.3% showed TERT copy number gain, and the incidence of this gain in acral melanoma (61.5%) was higher than that in other melanoma subtypes (P=0.02). The telomerase inhibitors 6-thio-2'-deoxyguanosine (6-Thio-dG) and epigallocatechin-3-gallate (EGCG) inhibited cell viability and repressed tumor growth in PDX models with TERT copy number gain. TERT copy number gain is frequently observed in Chinese patients with melanoma. Targeting telomerase may benefit melanoma patients with TERT copy number gain.

Telomerase activation in small intestinal neuroendocrine tumours is associated with aberrant TERT promoter methylation, but not hot-spot mutations

ABSTRACTTelomere maintenance is a critical requirement for enabling replicative immortality and tumour development. Here, telomerase expression and activity, telomere length (TL) and potential regulatory factors that can underlie telomerase machinery alterations in small intestinal neuroendocrine tumours (SI-NETs) were analyzed. Telomerase activity assessed by TRAP assay was increased in SI-NETs compared to normal ileum (P < 0.001). The telomerase reverse transcriptase gene (TERT) was over-expressed in SI-NETs vs. normal ileal samples (P = 0.01). Furthermore, relative TL assessed by qPCR was found shorter in tumours compared with normal ileum (P = 0.02) and in distant metastasis samples compared to primary tumours and local metastases (P= 0.02). TERT promoter hotspot mutations were not present and TERT copy number gain was only observed in 3/70 tumour samples. TERT or chromosome 18 copy number alterations were not associated with telomerase expression and activity or TL. However, hypermethylation of TERT promoter in Region B – in the proximity of the transcription start site – was inversely correlated with TERT expression and telomerase activity and positively correlated with TL. Global LINE1 methylation was positively correlated with TERT promoter Region B methylation and was inversely correlated with telomerase activity, TERT expression and the upstream Region A methylation. The results show that telomerase activation, TERT expression and shorter telomeres are commonly found in SI-NETs. Aberrant DNA methylation of TERT promoter and of LINE1 can be implicated in abnormal regulation of TERT in SI-NETs.

TERT copy gain predicts the outcome of high-dose interferon α-2b therapy in acral melanoma.

BackgroundAsian populations are more likely to develop acral melanoma (AM) than Caucasians. The high-dose interferon (HD-IFN) α-2b regimen is the main adjuvant treatment for AM. TERT encodes the catalytic subunit of telomerase reverse transcriptase, which plays an important role in melanoma. Frequent TERT mutation and increased TERT gene expression have been described in AM. Our study aimed to investigate the status and the clinical significance of TERT copy number in a large cohort of patients with AM and to analyze the relationship between TERT copy number gain and the efficiency of HD-IFN.Patients and methodsA total of 573 melanoma samples were retrospectively collected and analyzed for TERT copy number via Sanger sequencing. Clinical data of patients were also collected.ResultsTERT copy gain (copy number >2) was detected in 257 of the 573 patients with AM (44.9%). Of the 573 patients, 81 (14.1%) had a high copy gain (copy number >4). Patients with ulceration showed a significantly higher copy gain rate of TERT compared to the patients without ulceration (P=0.028). Patients with a tumor thicker than 4 mm also had a higher copy number rate of TERT than those with <4 mm (P=0.048). Our results showed that the overall survival (OS) was not significantly different between patients with and without TERT copy gain (P=0.890). However, among the 278 patients who received an HD-IFN regimen, Kaplan–Meier survival analysis demonstrated a significant correlation between TERT copy gain and relapse-free survival (RFS) (P=0.008). In addition, multivariate Cox regression assays validated TERT copy gain to be an independent prognostic factor of RFS for patients with AM undergoing HD-IFN therapy (hazard ratio =1.50; P=0.019).ConclusionThe copy number status of TERT might be a predictor for HD-IFN efficacy, but it is not a prognostic factor of OS in patients with AM.

Prognostic Value of TERT Alterations, Mutational and Copy Number Alterations Burden in Urothelial Carcinoma

Point mutations in the TERT gene promoter occur at high frequency in multiple cancers, including urothelial carcinoma (UC). However, the relationship between TERT promoter mutations and UC patient outcomes is unclear due to conflicting reports in the literature. In this study, we examined the association of TERT alterations, tumor mutational burden per megabase (Mb), and copy number alteration (CNA) burden with clinical parameters and their prognostic value in a cohort of 398 urothelial tumors. The majority of TERT mutations were located at two promoter region hotspots (chromosome 5, 1 295 228 C>T and 1 295 250 C>T). TERT alterations were more frequently present in bladder tumors than in upper tract tumors (73% vs 53%; p=0.001). ARID1A, PIK3CA, RB1, ERCC2, ERBB2, TSC1, CDKN1A, CDKN2A, CDKN2B, and PTPRD alterations showed significant co-occurrence with TERT alterations (all p<0.0025). TERT alterations and the mutational burden/Mb were independently associated with overall survival (hazard ratio[HR] 2.31, 95% confidence interval [CI] 1.46–3.65; p<0.001; and HR 0.96, 95% CI 0.93–0.99; p=0.002), disease-specific survival (HR 2.23, 95% CI 1.41–3.53; p<0.001; and HR 0.96, 95% CI 0.93–0.99; p=0.002), and metastasis-free survival (HR 1.63, 95% CI 1.05–2.53; p=0.029; and HR 0.98, 95% CI 0.96–1.00; p=0.063) in multivariate models. Patient summaryThe majority of TERT gene mutations that we detected in urothelial carcinoma are located at two promoter hotspots. Urothelial tumors with TERT alterations had worse prognosis compared to tumors without TERT alterations, whereas tumors with a higher mutational burden had more favorable outcome compared to tumors with low mutational burden.

Abstract 3389: Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma

Abstract Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. Contrasting with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis, and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM. Citation Format: Winnie S. Liang, William Hendricks, Jeffrey Kiefer, Jessica Schmidt, Shobana Sekar, John Carpten, David W. Craig, Jonathan Adkins, Lori Cuyugan, Zarko Manojlovic, Rebecca F. Halperin, Adrienne Helland, Sara Nasser, Christophe Legendre, Laurence H. Hurley, Karthigayini Sivaprakasam, Douglas B. Johnson, Holly Crandall, Klaus J. Busam, Victoria Zismann, Valerie De Luca, Jeeyun Lee, Aleksandar Sekulic, Charlotte E. Ariyan, Jeffrey Sosman, Jeffrey Trent. Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3389. doi:10.1158/1538-7445.AM2017-3389

Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma.

Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.

Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D.

Next‐generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large‐cell neuroendocrine carcinomas, and 33 small‐cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole‐exome sequencing and high‐coverage targeted sequencing of 418 genes. Eighty‐eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin‐remodelling genes, including those encoding histone modifiers and members of SWI–SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC (p = 0.0022). In conclusion, molecular profiling may complement histology for better diagnostic definition and prognostic stratification of LNETs. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Genetic and epigenetic background and protein expression profiles in relation to telomerase activation in medullary thyroid carcinoma.

Medullary thyroid carcinomas (MTCs) exhibit telomerase activation in strong association with shorter patient survival. To understand the background of telomerase activation we quantified TERT copy numbers and TERT promoter methylation in 42 MTCs and normal thyroid references. Gain of TERT was demonstrated by quantitative PCR in 5/39 sporadic MTC. Increased methylation index (MetI) for CpG methylation at the TERT promoter was found in sporadic MTCs (P < 0.0001) and in MEN 2 associated MTCs (P = 0.011) vs. normal thyroid tissues. MetI correlated positively with TERT gene expression (r = 0.432, P = 0.006) and negatively with telomere length (r = −0.343, P = 0.032). MTC cases with MetI above the median of 52% had shorter survival as compared to cases with lower MetI (P = 0.005 for overall survival and P = 0.007 for disease-related survival). Protein expression profiles obtained by mass spectrometry were then studied in relation to telomerase activation in MTCs. Comparing protein levels between tumors defined by telomerase activity status, 240 proteins were associated with telomerase activity. Among telomerase activation positive cases a set of proteins was found to discriminate between MTCs with high and low TERT gene expression with enrichment for proteins involved in telomerase regulation. XRCC5 mRNA expression was found increased in MTCs vs. normal thyroid (P = 0.007). In conclusion the findings suggest a role for TERT copy number gain, TERT promoter methylation and XRCC5 expression in telomerase activation and telomere maintenance of MTC.

TERT and AURKA Gene Copy Number Gains Enhance the Detection of Acral Lentiginous Melanomas by Fluorescence in Situ Hybridization

The study of specific chromosomal loci through fluorescence in situ hybridization (FISH) is useful in differential diagnosis of melanocytic tumors. However, sensitivity rates vary, probably because of molecular heterogeneity. Acral lentiginous melanomas are characterized by copy number gains of small genomic regions, including CCND1, TERT, and AURKA. In a series of 58 acral melanocytic lesions, we explored the value of a four-color FISH probe, used in addition to determining MYC gene status, and assessed the potential diagnostic usefulness of newly developed probes targeting TERT and AURKA. Moreover, we tested CCND1, TERT, and AURKA protein expression by immunohistochemistry. The four-color FISH probe detected 85.3% of melanomas and 29.4% of TERT and AURKA copy number gains. Sensitivity was 97% (confidence interval 95%, 82.9% to 99.8%) for the combined results of all probes. No MYC copy number gains were detected. No nevi showed aberrations. Immunohistochemistry revealed a higher percentage of cells positive for CCND1, TERT, and AURKA protein in melanomas than in nevi (P ≤ 0.001). A significant correlation between gene copy number gain and protein expression was found for CCND1 (P = 0.015). Our results indicate that addition of specific FISH probes to the current probe could improve sensitivity for the diagnosis of acral melanomas. Further studies in larger numbers of cases are needed to validate these results.

Recurrent pre‐existing and acquired DNA copy number alterations, including focal TERT gains, in neuroblastoma central nervous system metastases

Stage 4 neuroblastomas have a high rate of local and metastatic relapse and associated disease mortality. The central nervous system (CNS) is currently one of the most common isolated relapse sites, yet the genomic alterations that contribute to these metastases are unknown. This study sought to identify recurrent DNA copy number alterations (CNAs) and target genes relating to neuroblastoma CNS metastases by studying 19 pre-CNS primary tumors and 27 CNS metastases, including 12 matched pairs. SNP microarray analyses revealed that MYCN amplified (MYCNA) tumors had recurrent CNAs different from non-MYCNA cohorts. Several CNAs known to be prevalent among primary neuroblastomas occurred more frequently in CNS metastases, including 4p-, 7q+, 12q+, and 19q- in non-MYCNA metastases, and 9p- and 14q- irrespective of MYCNA status. In addition, novel CNS metastases-related CNAs included 18q22.1 gains in non-MYCNA pre-CNS primaries and 5p15.33 gains and 15q26.1→tel losses in non-MYCNA CNS metastases. Based on minimal common regions, gene expression, and biological properties, TERT (5p), NR2F2 (15q), ALDH1A3 (15q), CDKN2A (9p), and possibly CDH7 and CDH19 (18q) were candidate genes associated with the CNS metastatic process. Notably, the 5p15 minimal common region contained only TERT, and non-MYCNA CNS metastases with focal 5p15 gains had increased TERT expression, similar to MYCNA tumors. These findings suggest that a specific genomic lesion (18q22.1 gain) predisposes to CNS metastases and that distinct lesions are recurrently acquired during metastatic progression. Among the acquired lesions, increased TERT copy number and expression appears likely to function in lieu of MYCNA to promote CNS metastasis.

Frequent high telomerase reverse transcriptase expression in primary oral squamous cell carcinoma

Gene copy number gain of chromosomal arm 5p is frequently found in oral squamous cell carcinoma (OSCC) suggesting the activation of proto-oncogenes. TERT is a candidate gene encoding for human telomerase reverse transcriptase (hTERT). The aim of the present study was to elucidate the relevance of TERT copy number gain and high hTERT expression in OSCC. Fluorescence in situ hybridization (FISH) for TERT and immunohistochemistry (IHC) for hTERT were performed to analyze TERT copy numbers and hTERT expression, respectively, on tissue microarray (TMA) sections including n = 247 OSCC and n = 105 pharyngeal and laryngeal squamous cell carcinomas (PSCC/LSCC). Increased hTERT protein expression was more frequently found in OSCC (71.1%, 155/218) than in PSCC/LSCC (36.0%, 35/89) (P < 0.001). By contrast, specific TERT amplifications were less common in OSCC (2.1%, 4/191) compared with PSCC/LSCC (9.9%, 8/81) (P = 0.047). High hTERT expression is a frequent finding in OSCC. It might be a promising target for the development of specific anti-neoplastic therapy approaches.