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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., What's new? Telomerase reverse transcriptase (TERT) activation is central to cancer cell immortalization. It acts, however, through relatively unknown mechanisms. In urothelial bladder cancer (UBC) in particular, TERT activation can occur in the presence or absence of mutation, raising questions about alternative activation mechanisms. Our study shows that hypermethylation of the TERT promoter (THOR) plays a key part in UBC, being a dynamic and progressive process, with hypermethylation levels increasing with bladder cancer severity. Moreover, both hypermethylation and TERT promoter mutation contributed to increased telomerase expression. The findings provide insight into telomere biology in UBC and may be applicable to other tumors.

Background Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers. Main body The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation. hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies. Conclusion Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers. info:eu-repo/semantics/publishedVersion

Aim: We explore the biomarker potential of the TERT hypermethylated oncologic region (THOR) in pancreatic cancer. Materials & methods: We assessed the methylation status of THOR using the cancer genome atlas data on the cohort of pancreatic cancer (n = 193 patients). Results: THOR was significantly hypermethylated in pancreatic tumor tissue when compared with the normal tissue used as control (p < 0.0001). Also, THOR hypermethylation could distinguish early stage I disease from normal tissue and was associated with worse prognosis. Discussion: We found that THOR is hypermethylated in pancreatic tumor tissue when compared with normal tissue and that THOR methylation correlates with TERT expression in tumor samples. Conclusion: Our preliminary findings support the diagnostic and prognostic values of THOR in pancreatic cancer. FCT [PD/BD/105899/2014] LPCC-Fundacao PT fellowships CNPQ (Brazilian National Council for Science and Technology, Brasilia, DF, Brazil [402621/2016-6] info:eu-repo/semantics/publishedVersion

Replicative immortality is a hallmark of cancer cells governed by telomere maintenance. Approximately 90% of human cancers maintain their telomeres by activating telomerase, driven by the transcriptional upregulation of telomerase reverse transcriptase (TERT). Although TERT promoter mutations (TPMs) are a major cancer-associated genetic mechanism of TERT upregulation, many cancers exhibit TERT upregulation without TPMs. In this study, we describe the TERT hypermethylated oncological region (THOR), a 433-bp genomic region encompassing 52 CpG sites located immediately upstream of the TERT core promoter, as a cancer-associated epigenetic mechanism of TERT upregulation. Unmethylated THOR repressed TERT promoter activity regardless of TPM status, and hypermethylation of THOR counteracted this repressive function. THOR methylation analysis in 1,352 human tumors revealed frequent (>45%) cancer-associated DNA hypermethylation in 9 of 11 (82%) tumor types screened. Additionally, THOR hypermethylation, either independently or along with TPMs, accounted for how approximately 90% of human cancers can aberrantly activate telomerase. Thus, we propose that THOR hypermethylation is a prevalent telomerase-activating mechanism in cancer that can act independently of or in conjunction with TPMs, further supporting the utility of THOR hypermethylation as a prognostic biomarker.

// Pedro Castelo-Branco 1, 2, 3, * , Ricardo Leao 1, 4, 5, * , Tatiana Lipman 1 , Brittany Campbell 1 , Donghyun Lee 1 , Aryeh Price 1 , Cindy Zhang 1 , Abolfazl Heidari 1 , Derek Stephens 1 , Stefan Boerno 6 , Hugo Coelho 5 , Ana Gomes 5 , Celia Domingos 2, 3 , Joana D. Apolonio 2, 3 , Georg Schafer 11 , Robert G. Bristow 7 , Michal R. Schweiger 8, 9 , Robert Hamilton 4 , Alexandre Zlotta 4, 10 , Arnaldo Figueiredo 5 , Helmut Klocker 11 , Holger Sultmann 12 , Uri Tabori 1 1 Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada 2 Regenerative Medicine Program, Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal 3 Centre for Biomedical Research (CBMR), University of Algarve, Faro, Portugal 4 Division of Urology, Department of Surgical Oncology Princess Margaret Cancer Center, University of Toronto, Toronto, Ontario, Canada 5 Servico de Urologia e Transplantacao Renal, Centro Hospitalar Universitario Coimbra EPE, Faculty of Medicine, University of Coimbra, Coimbra, Portugal 6 Sequencing Core Facility, Max Planck Institute for Molecular Genetics, Berlin, Germany 7 Department of Radiation Oncology, Princess Margaret Cancer Center, Toronto, Ontario, Canada 8 Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany 9 Cologne Center for Genomics, Cologne University, Cologne, Germany 10 Division of Urology, Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada 11 Department of Urology, Medical University of Innsbruck, Innsbruck, Austria 12 Cancer Genome Research, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany * These authors contributed equally to this work Correspondence to: Uri Tabori, email: uri.tabori@sickkids.ca Pedro Castelo-Branco, email: pjbranco@ualg.pt Keywords: TERT, prostate cancer, biomarker, diagnostic, Gleason score Received: March 31, 2016 Accepted: June 30, 2016 Published: July 16, 2016 ABSTRACT The identification of new biomarkers to differentiate between indolent and aggressive prostate tumors is an important unmet need. We examined the role of THOR ( TERT Hypermethylated Oncological Region) as a diagnostic and prognostic biomarker in prostate cancer (PCa). We analyzed THOR in common cancers using genome-wide methylation arrays. Methylation status of the whole TERT gene in benign and malignant prostate samples was determined by MeDIP-Seq. The prognostic role of THOR in PCa was assessed by pyrosequencing on discovery and validation cohorts from patients who underwent radical prostatectomy with long-term follow-up data. Most cancers ( n = 3056) including PCa ( n = 300) exhibited hypermethylation of THOR. THOR was the only region within the TERT gene that is differentially methylated between normal and malignant prostate tissue ( p < 0.0001). Also, THOR was significantly hypermethylated in PCa when compared to paired benign tissues ( n = 164, p < 0.0001). THOR hypermethylation correlated with Gleason scores and was associated with tumor invasiveness ( p = 0.0147). Five years biochemical progression free survival (BPFS) for PCa patients in the discovery cohort was 87% (95% CI 73–100) and 65% (95% CI 52–78) for THOR non-hypermethylated and hypermethylated cancers respectively ( p = 0.01). Similar differences in BPFS were noted in the validation cohort ( p = 0.03). Importantly, THOR was able to predict outcome in the challenging (Gleason 6 and 7 (3 + 4)) PCa ( p = 0.007). For this group, THOR was an independent risk factor for BPFS with a hazard-ratio of 3.685 ( p = 0.0247). Finally, THOR hypermethylation more than doubled the risk of recurrence across all PSA levels (OR 2.5, p = 0.02).

© The Author(s). 2019. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated., Background: Breast cancer is a highly heterogeneous disease resulting in diverse clinical behaviours and therapeutic responses. DNA methylation is a major epigenetic alteration that is commonly perturbed in cancers. The aim of this study is to characterize the relationship between DNA methylation and aberrant gene expression in breast cancer. Methods: We analysed DNA methylation and gene expression profiles from breast cancer tissue and matched normal tissue in The Cancer Genome Atlas (TCGA). Genome-wide differential methylation analysis and methylation-gene expression correlation was performed. Gene expression changes were subsequently validated in the METABRIC dataset. The Oncoscore tool was used to identify genes that had previously been associated with cancer in the literature. A subset of genes that had not previously been studied in cancer was chosen for further analysis. Results: We identified 368 CpGs that were differentially methylated between tumor and normal breast tissue (∆β > 0.4). Hypermethylated CpGs were overrepresented in tumor tissue and were found predominantly (56%) in upstream promoter regions. Conversely, hypomethylated CpG sites were found primarily in the gene body (66%). Expression analysis revealed that 209 of the differentially-methylated CpGs were located in 169 genes that were differently expressed between normal and breast tumor tissue. Methylation-expression correlations were predominantly negative (70%) for promoter CpG sites and positive (74%) for gene body CpG sites. Among these differentially-methylated and differentially-expressed genes, we identified 7 that had not previously been studied in any form of cancer. Three of these, TDRD10, PRAC2 and TMEM132C, contained CpG sites that showed diagnostic and prognostic value in breast cancer, particularly in estrogen-receptor (ER)-positive samples. A pan-cancer analysis confirmed differential expression of these genes together with diagnostic and prognostic value of their respective CpG sites in multiple cancer types. Conclusion: We have identified 368 DNA methylation changes that characterize breast cancer tumor tissue, of which 209 are associated with genes that are differentially-expressed in the same samples. Novel DNA methylation markers were identified, of which cg12374721 (PRAC2), cg18081940 (TDRD10) and cg04475027 (TMEM132C) show promise as diagnostic and prognostic markers in breast cancer as well as other cancer types., This work was supported by the research grant UID/BIM/04773/2013 CBMR from FCT and Maratona da Saúde. JA is supported by PD/BD/105899/2014 FCT fellowship.