Telomerase Reverse Transcriptase Promoter Methylation Research Articles

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.

P13.01 TERTpromoter methylation is significantly associated withTERTupregulation and tumor progression in pituitary adenomas

Abstract BACKGROUND Activation of telomerase plays a critical role in tumor development. Somatic alterations in the promoter of the telomerase reverse transcriptase (TERT) gene are a major mechanism of upregulating telomerase. Several mechanisms have been associated with TERT abnormalities, including TERT promoter mutations or methylation. Mutations in the TERT promoter have been observed in a subset of brain tumors, especially in adult gliomas. In pituitary adenomas (PAs), however, TERT abnormalities are not fully understood. The present study aimed to investigate not only mutational but also methylation status changes in the TERT promoter in PAs and to analyze their correlations with clinical variables. MATERIAL AND METHODS We retrospectively studied 70 PAs consisting of 53 primary and 17 recurrent samples. Clinical data, including age at surgery, sex, tumor size, tumor subtype, resection rate, presence or absence of postoperative irradiation, and progression-free survival (PFS), were obtained from medical records. First, we investigated TERT promoter hotspot mutations via Sanger sequencing. Next, we quantified the methylation status of the TERT promoter using methylation-sensitive high-resolution melting analysis (MS-HRM). Finally, we investigated TERT mRNA expression levels using real-time quantitative PCR. Fisher’s exact test was applied to evaluate the statistical significance between TERT promoter methylation status and tumor recurrence. PFS was calculated using Kaplan-Meier estimates and compared between methylated with ummethylated PAs with the log-rank test. The correlation between TERT promoter methylation status and mRNA levels was analyzed with the Mann-Whitney U-test. PFS was analyzed using multivariate analysis with the Cox proportional hazards model and included the following variables: age, sex, tumor size, tumor subtype, resection rate, radiation therapy, and methylation status. RESULTS TERT promoter hotspot mutations were not observed in any PA sample. Nineteen percent of PAs exhibitedTERT promoter methylation, which was significantly predominant in recurrent PA samples. PFS was significantly shorter in the methylated cases than in the unmethylated cases. Higher TERT expression levels were correlated with methylation status. CONCLUSION We found that TERT promoter methylation upregulated TERT expression and was associated with shorter PFS in PAs. Our results suggest thatTERT promoter methylation may be a potential biomarker for predicting tumor recurrence in PAs.

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.

PF540 TERT PROMOTER METHYLATION AS A POTENTIAL PROGNOSTIC BIOMARKER FOR PROGRESSION OF UNEXPLAINED CYTOPENIA TO MDS AND AML

Background:Patients with myelodysplastic syndrome (MDS) often have mutations in epigenetic regulators and altered DNA methylation in their blood cells. Patients with unexplained cytopenia that do not fulfill the criteria for MDS are referred to as having idiopathic cytopenia of undetermined significance (ICUS) or, if they have MDS‐related mutations, clonal cytopenia of undetermined significance (CCUS).A previous study (Zhao et al. BJH 2016) showed that acute myeloid leukemia (AML) patients with preceding MDS (MDS/AML) have hypermethylation of the telomerase reverse transcriptase (TERT) promoter compared to healthy controls. Increased TERT promoter methylation may cause impaired telomerase activity and thereby shortening of telomeres and increased risk of progression to AML.Aims:We aimed to investigate DNA methylation of the TERT promoter to evaluate its potential as a biomarker of disease severity in patients with unexplained cytopenia and MDS.Methods:TERT promoter methylation of two regions (region 1 covering 5 CpG sites 456 to 424 bp upstream of the TSS and region 2 covering 6 CpG sites 384 to 328 bp upstream of the TSS) was analyzed by pyrosequencing in a cohort of 185 patients with ICUS (n = 65), CCUS (n = 49) or MDS (n = 71). Results are presented as DNA methylation percentage of region 1 and 2 (mean ± SD).Results:The 185 included patients (111 men, 74 women) had a median age of 69 years (ICUS: 65 years, CCUS: 70 years, MDS: 72 years) and the 15 healthy controls (6 men, 7 women) had a median age of 57 years.One‐way ANOVA analysis revealed significantly differences in TERT promoter methylation between all groups for both regions (region 1: p = 0.0004, region 2: p = 7.4 × 10–5).TERT promoter methylation in region 1 was significantly higher in all disease groups compared to healthy controls, with increasing methylation and significance levels with increased disease severity (ICUS: 8.3 ± 4.8, p = 0.03; CCUS: 10.2 ± 7.2, p = 0.0007, MDS: 11.3 ± 7.7, p = 1.9 × 10–6; Figure 1A). Furthermore, TERT promoter methylation levels of both CCUS and MDS patients were significantly higher than ICUS patients (p = 0.02 and p = 1.8 × 10–6, respectively.In region 2, MDS patients had significantly higher methylation (MDS: 8.8 ± 8.1, p = 0.001) compared to healthy controls (5.4 ± 3.2). TERT promoter methylation of region 2 in ICUS and CCUS patients was not significantly different from healthy controls or MDS patients (Figure 1B).Figure 1: Box‐and‐whisper plots of TERT promoter methylation percentage in region 1 (A) and region 2 (B) in 185 ICUS, CCUS, MDS patients and 15 age‐matched healthy controls.Summary/Conclusion:Promoter methylation of TERT increases gradually from healthy controls to ICUS, CCUS and MDS patients. Our study suggests that DNA methylation of the TERT promoter reflects disease severity of unexplained cytopenia and MDS patients. We are currently investigating if TERT promoter methylation can be used as a biomarker for survival and progression to MDS and AML.image

Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines.

Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.

TERT promoter methylation is significantly associated with TERT upregulation and disease progression in pituitary adenomas.

Alterations in the promoter of the telomerase reverse transcriptase (TERT) gene are a major mechanism of upregulating telomerase, which plays a crucial role in tumor development. Mutations in the TERT promoter have been observed in a subset of brain tumors, including adult gliomas and high-grade meningiomas. In pituitary adenomas (PAs), however, abnormalities in TERT are not fully understood. The present study aimed to investigate not only mutational but also methylation changes in the TERT promoter in PAs and to analyze their correlations with clinical variables. We retrospectively studied 70 PAs consisting of 53 primary and 17 recurrent samples. Clinical data, including age at surgery, sex, largest tumor dimension, tumor subtype, resection rate, and progression-free survival (PFS), were obtained from medical records. We investigated TERT promoter hotspot mutations via Sanger sequencing and quantified the methylation status of the TERT promoter using methylation-sensitive high-resolution melting analysis (MS-HRM). Additionally, we investigated TERT mRNA expression using real-time quantitative PCR. TERT promoter hotspot mutations were not observed in any PA sample, while 16% of PAs exhibited TERT promoter methylation. PAs with methylated TERT promoters were significantly more likely to show disease progression, shorter PFS, and higher TERT expression levels compared to those with unmethylated promoters. This is the first study showing that TERT promoter methylation is associated with disease progression and shorter PFS as well as upregulated TERT expression in PAs. Our results suggest that TERT promoter methylation may be a potential biomarker for predicting tumor recurrence in PAs.

Abstract 5329: Telomerase reverse transcriptase promoter methylation in metastatic melanoma

Abstract Aberrant methylation of a particular region of the Telomerase Reverse Transcriptase (TERT) promoter, termed the upstream of transcription start site (UTSS), is associated with increased TERT expression and may be useful for differentiating malignant tumors, such as childhood brain tumors, prostate cancer, gastric cancer and melanoma, from their benign/low-grade histologic mimics. However, it is unclear whether UTSS methylation is actually responsible for transcription or is only associated with it. The purpose of this study was to test the hypothesis that UTSS methylation is an activating mechanism for transcription of this gene in melanoma. Since the majority of melanomas express TERT mRNA, we predicted that i) the majority of these samples would have some detectable aberration in the TERT promoter that might account for expression and ii) UTSS methylation would preferentially occur in samples which lacked other recurrent TERT promoter aberrations (point mutation or rearrangement). To test this prediction, we used a cohort of 57 adult melanoma samples to catalog the presence of UTSS methylation (by high-throughput bisulfite-sequencing), promoter mutation (by Sanger sequencing) and/or promoter rearrangement (by fluorescence in situ hybridization) in each case. 5 cases of atypical Spitz tumors and congenital nevi served as negative controls, since these do not express TERT mRNA and were therefore not expected to have such aberrations. As predicted, 9 of the 10 melanoma samples which lacked promoter mutations or rearrangement had a high level of UTSS methylation. Unexpectedly, UTSS methylation sometimes overlapped with the presence of point mutations (9 of 40 cases) or promoter rearrangement (3 of 5 cases). None of the negative control samples expressed TERT mRNA or harbored any promoter aberrations. We next predicted that, in samples with methylated UTSS, the actively transcribed TERT allele(s) would harbor the methylated UTSS while the transcriptionally silent allele(s) would not. Since methylation analysis of clinical samples is complicated by the presence of unmethylated UTSS from contaminating normal tissue, we tested this prediction in two relatively pure cancer cell lines. Although only one TERT allele was transcribed in each cell line, as measured by digital droplet PCR, we found that in each case both the transcriptionally active and transcriptionally silent alleles had methylated UTSS. These results suggest that UTSS methylation is not sufficient for TERT upregulation, although it may still be useful as a biomarker for diagnosing melanoma and other cancers. Future studies will be aimed to determine if methylation of other regions of the TERT promoter marks the active allele in samples lacking mutations or rearrangement. Citation Format: Sumit Borah, Seung J. Lee, Yiping Fan, Raymond L. Barnhill, Reinhard Dummer, John M. Kirkwood, Armita Bahrami. Telomerase reverse transcriptase promoter methylation in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5329.

TERT promoter hypermethylation is associated with poor prognosis in adrenocortical carcinoma.

Telomere maintenance, most commonly achieved by telomerase activation through induction of the telomerase reverse transcriptase (TERT) gene, is required for cell immortalization, a hallmark of cancer. Adrenocortical carcinoma (ACC) is an endocrine tumor for which TERT promoter mutations and telomerase activation have been reported. The present study assessed alterations of the TERT gene locus and telomere length in relation to clinical characteristics in ACC. In total, 38 cases of ACC with known TERT promoter mutational status were included. TERT promoter methylation densities were assessed by pyrosequencing, and TERT copy numbers and telomere length were determined by quantitative polymerase chain reaction analysis, followed by comparison of the mRNA expression of TERT and clinical parameters. The ACC tissue samples showed increased TERT copy numbers, compared with normal adrenal tissue (NAT) samples (P=0.001). Mutually exclusive TERT copy number gains or promoter mutation were present in 70% of the ACC samples. The ACC tissues exhibited higher levels of CpG promoter methylation of all eight CpG sites investigated within the ‑578 to ‑541 bp (Region A), compared with the NATs (P=0.001). High methylation density at this region was associated with metastatic disease and/or relapse, poor survival rates and higher European Network for the Study of Adrenal Tumor stage (P<0.05). The mRNA expression of TERT was inversely correlated with methylation density at ‑162 to ‑100 bp (Region B). Correlation was observed between relative telomere length and the gene expression of TERT. It was concluded that epigenetic alterations of the TERT promoter are frequent and associated with advanced disease and poorer clinical outcome in ACC.

De novo and secondary anaplastic meningiomas: a study of clinical and histomolecular prognostic factors.

Following recent studies underlining the differences between de novo and secondary anaplastic meningiomas and the prognostic value of telomerase reverse transcriptase (TERT) promoter mutation, we decided to conduct a multicenter retrospective study to address these questions and determine specific prognostic factors in each of these 2 anaplastic meningioma subgroups. Among the 68 meningioma cases initially selected, only 57 were confirmed as anaplastic meningiomas after centralized pathological review. TERT promoter mutation analysis was performed in all cases. Median overall survival was 2.6 years and 5-year survival rate was 10%. This study confirmed the better prognosis of de novo anaplastic meningiomas (28 tumors) compared with secondary anaplastic meningiomas (29 tumors) (P = 0.02). In the "de novo" group, meningiomas diagnosed on histological anaplasia alone had a better prognosis than those in patients with a high number of mitoses with or without anaplasia (P = 0.01). In the "secondary" group, tumors demonstrate very heterogeneous clinical courses leading to malignant transformation, and time to first relapse as a low-grade tumor was a strong predictor of overall survival (P = 0.0007). TERT promoter mutation in anaplastic meningiomas was rare (14%) and did not influence overall survival but was associated with a shorter recurrence-free survival in the secondary anaplastic meningioma subgroup (P = 0.02). The absence of TERT promoter methylation, although rare (3/33 cases), may be associated with prolonged overall survival (P = 0.02). This study highlights the different prognoses of de novo and secondary anaplastic meningiomas with specific prognostic factors in each subgroup. The analysis of TERT mutation and methylation could provide additional prognostic insights.

TERT Promoter Hypermethylation in Gastrointestinal Cancer: A Potential Stool Biomarker.

There is a high demand for noninvasive screening tools for gastrointestinal cancer (GIC) detection, and GIC-specific markers are required for such purposes. It is established that induction of the telomerase reverse transcriptase gene (TERT) coupled with telomerase activation is essential for cancer development/progression and aberrant TERT promoter methylation of specific 5'-C-phosphate-G-3' (CpGs) has been linked to TERT induction in oncogenesis. Here we analyzed TERT promoter methylation in fecal samples from GIC patients and healthy adults and determined its value as a stool biomarker for GIC detection. Sixty-nine GIC patients (34 colorectal carcinoma and 35 gastric cancer) and 62 healthy adults were recruited and fecal samples were collected. Paired tumors and adjacent non-cancerous tissues from 34 patients and normal mucosa tissues from 12 healthy individuals were collected. TERT promoter methylation density was determined using pyrosequencing. We identified two GIC-specific methylation sites at -218 (CpG site 1) and -210 (CpG site 2) in the TERT promoter in tumor tissues. Methylated TERT promoter CpG sites 1 and 2 were also detectable in patient stool, while only background levels were observed in healthy individuals. The overall sensitivity reached 52.2% (95% confidence interval [CI]: 48.3-56.0) for fecal methylated TERT promoter assays at 90% specificity, which was comparable to other known stool methylation markers for GIC detection. The combined assays of fecal TERT promoter methylation and occult blood (OB) significantly improved sensitivity and specificity in colorectal cancer (area under curves for methylation alone: 0.798, 95% CI: 0.707-0.889 vs. methylation + OB: 0.920, 95% CI: 0.859-0.981; p = .028), but not in gastric cancer. This proof-of-concept study suggests the feasibility of stool TERT promoter methylation analyses as an additional tool in noninvasive GIC screening. Induction of telomerase reverse transcriptase (TERT) expression coupled with telomerase activation is essential for cancer development/progression, while aberrant TERT promoter methylation has been linked to TERT induction in oncogenesis. We identified two cancer-specific methylation sites (CpG1 and 2) in the TERT promoter in tumors from GIC patients. Methylated TERT promoter CpG sites 1 and 2 were detectable in patient stool, while only background levels were observed in healthy individuals. The sensitivity and specificity was comparable to other known stool methylation markers for GIC detection. This proof-of-concept study suggests the feasibility of stool TERT promoter methylation analyses for noninvasive screening of GIC.

Genetic and Epigenetic Alterations of TERT Are Associated with Inferior Outcome in Adolescent and Young Adult Patients with Melanoma

Progression of melanoma to distant sites in adolescents and young adults (AYAs) is not reliably predicted by clinicopathologic criteria. TERT promoter mutations when combined with BRAF/NRAS mutations correlate with adverse outcome in adult melanoma. To determine the prognostic value of TERT alterations in AYA melanoma, we investigated the association of TERT promoter mutations, as well as promoter methylation, an epigenetic alteration also linked to TERT upregulation, with TERT mRNA expression and outcome using a well-characterized cohort of 27 patients with melanoma (ages 8–25, mean 20). TERT mRNA expression levels were significantly higher in tumors harboring TERT promoter mutation and/or hypermethylation than those without either aberration (P = 0.046). TERT promoter mutations alone did not predict adverse outcomes (P = 0.50), but the presence of TERT promoter methylation, alone or concurrent with promoter mutations, correlated with reduced recurrence-free survival (P = 0.001). These data suggest that genetic and epigenetic alterations of TERT are associated with TERT upregulation and may predict clinical outcomes in AYA melanoma. A more exhaustive understanding of the different molecular mechanisms leading to increased TERT expression may guide development of prognostic assays to stratify AYA melanoma patients according to clinical risk.

Detection of Aberrant TERT Promoter Methylation by Combined Bisulfite Restriction Enzyme Analysis for Cancer Diagnosis

Aberrant CpG dinucleotide methylation in a specific region of the telomerase reverse transcriptase (TERT) promoter is associated with increased TERT mRNA levels and malignancy in several cancer types. However, routine screening of this region to aid cancer diagnosis can be challenging because i) several established methylation assays may inaccurately report on hypermethylation of this particular region, ii) interpreting the results of methylation assays can sometimes be difficult for clinical laboratories, and iii) use of high-throughput methylation assays for a few patient samples can be cost prohibitive. Herein, we describe the use of combined bisulfite restriction enzyme analysis (COBRA) as a diagnostic tool for detecting the hypermethylated TERT promoter using invitro methylated and unmethylated genomic DNA as well as genomic DNA from four melanomas and two benign melanocytic lesions. We compare COBRA with MassARRAY, a more commonly used high-throughput approach, in screening for promoter hypermethylation in 28 formalin-fixed, paraffin-embedded neuroblastoma samples. COBRA sensitively and specifically detected samples with hypermethylated TERT promoter and was as effective as MassARRAY at differentiating high-risk from benign or low-risk tumors. This study demonstrates the utility of this low-cost, technically straightforward, and easily interpretable assay for cancer diagnosis in tumors of an ambiguous nature.

Systematic analysis of telomere length and somatic alterations in 31 cancer types.

Cancer cells survive cellular crisis through telomere maintenance mechanisms. We report telomere lengths in 18,430 samples, including tumors and non-neoplastic samples, across 31 cancer types. Telomeres were shorter in tumors than in normal tissues and longer in sarcomas and gliomas than in other cancers. Among 6,835 cancers, 73% expressed telomerase reverse transcriptase (TERT), which was associated with TERT point mutations, rearrangements, DNA amplifications and transcript fusions and predictive of telomerase activity. TERT promoter methylation provided an additional deregulatory TERT expression mechanism. Five percent of cases, characterized by undetectable TERT expression and alterations in ATRX or DAXX, demonstrated elongated telomeres and increased telomeric repeat-containing RNA (TERRA). The remaining 22% of tumors neither expressed TERT nor harbored alterations in ATRX or DAXX. In this group, telomere length positively correlated with TP53 and RB1 mutations. Our analysis integrates TERT abnormalities, telomerase activity and genomic alterations with telomere length in cancer.

Tianshengyuan-1 (TSY-1) regulates cellular Telomerase activity by methylation of TERT promoter.

Telomere and Telomerase have recently been explored as anti-aging and anti-cancer drug targets with only limited success. Previously we showed that the Chinese herbal medicine Tianshengyuan-1 (TSY-1), an agent used to treat bone marrow deficiency, has a profound effect on stimulating Telomerase activity in hematopoietic cells. Here, the mechanism of TSY-1 on cellular Telomerase activity was further investigated using HL60, a promyelocytic leukemia cell line, normal peripheral blood mononuclear cells, and CD34+ hematopoietic stem cells derived from umbilical cord blood. TSY-1 increases Telomerase activity in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells with innately low Telomerase activity but decreases Telomerase activity in HL60 cells with high intrinsic Telomerase activity, both in a dose-response manner. Gene profiling analysis identified Telomerase reverse transcriptase (TERT) as the potential target gene associated with the TSY-1 effect, which was verified by both RT-PCR and western blot analysis. The β-galactosidase reporter staining assay showed that the effect of TSY-1 on Telomerase activity correlates with cell senescence. TSY-1 induced hypomethylation within TERT core promoter in HL60 cells but induced hypermethylation within TERT core promoter in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells. TSY-1 appears to affect the Telomerase activity in different cell lines differently and the effect is associated with TERT expression, possibly via the methylation of TERT promoter.

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.

Telomerase reverse transcriptase methylation predicts lymph node metastasis and prognosis in patients with gastric cancer.

PurposeTelomerase activity is associated with cellular immortalization and is present in most human tumors but absent in normal tissues. The activity of telomerase reverse transcriptase (TERT), a catalytic telomerase subunit, correlates with telomerase activity in tumors. The objective of this study was to investigate TERT promoter methylation and its prognostic impact in gastric cancer (GC).Patients and methodsThe analysis of TERT promoter methylation was performed in tumors and corresponding normal tissues of 116 patients with GC using a highly sensitive Sequenom Epityper assay. The expression of TERT in GC tissues was measured by quantitative real-time polymerase chain reaction.ResultsThe levels of TERT promoter methylation in GC samples were significantly higher than in normal adjacent tissues (P=0.002). Hypermethylation of TERT promoter was associated with high T-stage (P=0.024), late N-stage (P=0.006), and lymphovascular/neural invasion (P=0.035), without correlation with age, sex, or histological grade. Simple linear regression analysis showed that TERT mRNA correlated positively with TERT methylation (R2=0.562, P=0.001). Also, higher TERT mRNA expression was related to hypermethylation of TERT promoter in GC samples (P=0.005). Univariate analysis demonstrated that N-stage (P=0.002) and TERT promoter methylation (P=0.004) were predictive of overall survival. Furthermore, multivariate analysis confirmed that N-stage (P=0.013) and TERT promoter methylation (P=0.031) were independent prognostic indicators for overall survival.ConclusionOur data suggested that hypermethylation of TERT promoter may contribute to gastric wall invasion, lymph node metastasis, lymphovascular/neural invasion, and poor prognosis in GC. GC patients with hypermethylation of TERT promoter could be eligible for close follow-up.

The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer.

A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

Differential timing of oxidative DNA damage and telomere shortening in hepatitis C and B virus-related liver carcinogenesis.

In viral hepatitis, inflammation is correlated with chronic oxidative stress, one of the biological events leading to DNA damage and hepatocellular carcinoma (HCC) development. Aim of this study was to investigate the complex molecular network linking oxidative damage to telomere length and telomerase activity and regulation in hepatitis C and B virus-related liver carcinogenesis. We investigated 142 patients: 21 with HCC (in both tumor and peritumor tissues) and 121 with chronic viral hepatitis in different stages. We evaluated 8-hydroxydeoxyguanosine (8-OHdG), marker of oxidative DNA damage, OGG1 gene polymorphism, telomere length, telomerase activity, TERT promoter methylation, and mitochondrial TERT localization. In hepatitis C-related damage, 8-OHdG levels increased since the early disease stages, whereas hepatitis B-related liver disease was characterized by a later and sharper 8-OHdG accumulation (P=0.005). In C virus-infected patients, telomeres were shorter (P=0.03), whereas telomerase activity was higher in tumors than that in the less advanced stages of disease in both groups (P=0.0001, P=0.05), with an earlier increase in hepatitis C. Similarly, TERT promoter methylation was higher in tumor and peritumor tissues in both groups (P=0.02, P=0.0001). Finally, TERT was localized in mitochondria in tumor and peritumor samples, with 8-OHdG levels significantly lower in mitochondrial than those in genomic DNA (P=0.0003). These data describe a pathway in which oxidative DNA damage accumulates in correspondence with telomere shortening, telomerase activation, and TERT promoter methylation with a different time course in hepatitis B and C virus-related liver carcinogenesis. Finally, TERT localizes in mitochondria in HCC, where it lacks a canonical function.

Telomerase reverse transcriptase promoter methylation is related to a risk of recurrence in hepatocellular carcinoma

Supported by the Korea Healthcare Technology Research and Development Project of the Ministry of Health and Welfare of the Republic of Korea (HI12C1437). Potential conflict of interest: Nothing to report. To the Editor: We read with great interest the article published in this journal by Nault et al.,1 who observed 0% frequency of somatic mutations in the telomerase reverse transcriptase (TERT) promoter in cirrhotic livers, 6% in low‐grade dysplastic nodules, 19% in high‐grade dysplastic nodules, and 61% in early hepatocellular carcinoma (HCC). These data indicate that TERT promoter mutations are early prognostic markers for HCC, based on their increased frequency in liver carcinogenesis; such an effect was not observed in 10 cancer genes (CTNNB1, TP53, ARID1A, ARID2, NFE2L2, AXIN1, PIK3CA, KEAP1, RPS6KA3, and CDKN2A).1 Nault et al.1 demonstrated that TERT promoter mutations significantly contribute to increased TERT expression in liver carcinogenesis; here, we suggest an additional mechanism regulating TERT expression. Promoter methylation of TERT is observed in various human tumor tissues and transformed cell lines,2 and its hypermethylation is positively associated with TERT expression and poor prognosis in childhood brain tumors.3 Our study of 78 Korean HCC patients reinforces the possibility that TERT promoter methylation is involved in liver carcinogenesis. First, TERT messenger RNA expression was significantly higher in HCC tumor tissues possessing a methylated TERT promoter than in those possessing an unmethylated promoter (Mann‐Whitney test, P < 0.001; Fig. 1A). Second, TERT promoter methylation was associated with poor recurrence‐free survival in HCC patients (log‐rank test, P = 0.026; Fig. 1B) and a significant risk of recurrence, as evidenced by a hazard ratio of 4.612 (95% confidence interval = 1.048‐20.3, P = 0.0432). Because TERT promoter methylation was not associated with somatic mutations at the promoter in HCC tumors (chi‐squared test, P = 0.154), these two factors independently regulate TERT expression. Taken together, these results are consistent with those of previous studies,2 indicating that TERT promoter methylation is positively associated with TERT expression.Figure 1: Methylation at the TERT promoter correlates with high TERT expression and poor recurrence‐free survival rate. (A) The TERT expression level in HCC tumor tissues possessing unmethylated or methylated TERT promoters. Methylation status was checked by methylation‐specific polymerase chain reaction using primer pairs specific for unmethylated or methylated TERT promoters (the region is from 270 bp upstream of the ATG start site to 31 bp upstream of the ATG start site2). Boxes show median, 25th percentile, and 75th percentile, while whiskers show 5th and 95th percentiles. (B) Recurrence‐free survival rates of HCC patients. Recurrence‐free survival rates were estimated using the Kaplan‐Meier method, and the differences in survival rates were compared using the log‐rank test.Findings of this study and that by Nault et al.1 demonstrate that increased TERT expression contributes substantially to HCC progression, suggesting the potential of therapeutic strategies aimed at suppressing TERT expression. Acknowledgment The biospecimens for this study were provided by Seoul St. Mary's Hospital of the Catholic University of Korea and the Korea University Guro Hospital of National Biobank, a member of the National Biobank of Korea.

422 TERT promoter methylation is a pan-cancer biomarker with prognostic significance in prostate cancer patients

422 TERT promoter methylation is a pan-cancer biomarker with prognostic significance in prostate cancer patients