The TERT Promoter: A Key Player in the Fight for Cancer Cell Immortality.

The review describes the role of telomeres and telomerase in tumor progression, as well as various mechanisms of the activation of telomerase reverse transcriptase (TERT) expression in CNS tumors and other cancers. The main mechanism of TERT activation involves acquisition of somatic mutations by the TERT gene promoter (TERTp). The article presents information on the TERTp structure and transcription factors directly interacting with TERTp and regulating its transcription. The prospects of using the mutational status of TERTp as a prognostic marker of CNS malignancies and other tumors with a common profile of TERTp mutations are discussed.

Development of a Sensitive Digital Droplet PCR Screening Assay for the Detection of GPR126 Non-Coding Mutations in Bladder Cancer Urine Liquid Biopsies.

Recent whole-genome sequencing studies identified two novel recurrent mutations in the enhancer region of GPR126 in urothelial bladder cancer (UBC) tumor samples. This mutational hotspot is the second most common after the TERT promoter in UBC. The aim of the study was to develop a digital droplet PCR screening assay for the simultaneous detection of GPR126 mutations in a single tube. Its performance combined with TERT promoter mutation analysis was evaluated in urine of healthy volunteers (n = 50) and patients with cystitis (n = 22) and UBC (n = 70). The developed assay was validated using DNA constructs carrying the studied variants. None of the mutations were detected in control and cystitis group samples. GPR126 mutations were observed in the urine of 25/70 UBC patients (area under the ROC curve (AUC) of 0.679; mutant allele fraction (MAF) of 21.61 [8.30-44.52] %); TERT mutations-in 40/70 (AUC of 0.786; MAF = 28.29 [19.03-38.08] %); ≥1 mutation-in 47/70 (AUC of 0.836)). The simultaneous presence of GPR126 and TERT mutations was observed in 18/70 cases, with no difference in MAFs for the paired samples (31.96 [14.78-47.49] % vs. 27.13 [17.00-37.62] %, p = 0.349, respectively). The combined analysis of these common non-coding mutations in urine allows the sensitive and non-invasive detection of UBC.

Detection of TERT Promoter Mutations as a Prognostic Biomarker in Gliomas: Methodology, Prospects, and Advances.

This article reviews the existing approaches to determining the TERT promoter mutational status in patients with various tumoral diseases of the central nervous system. The operational characteristics of the most common methods and their transferability in medical practice for the selection or monitoring of personalized treatments based on the TERT status and other related molecular biomarkers in patients with the most common tumors, such as glioblastoma, oligodendroglioma, and astrocytoma, are compared. The inclusion of new molecular markers in the course of CNS clinical management requires their rapid and reliable assessment. Availability of molecular evaluation of gliomas facilitates timely decisions regarding patient follow-up with the selection of the most appropriate treatment protocols. Significant progress in the inclusion of molecular biomarkers for their subsequent clinical application has been made since 2016 when the WHO CNS classification first used molecular markers to classify gliomas. In this review, we consider the methodological approaches used to determine mutations in the promoter region of the TERT gene in tumors of the central nervous system. In addition to classical molecular genetical methods, other methods for determining TERT mutations based on mass spectrometry, magnetic resonance imaging, next-generation sequencing, and nanopore sequencing are reviewed with an assessment of advantages and disadvantages. Beyond that, noninvasive diagnostic methods based on the determination of the mutational status of the TERT promoter are discussed.

Urine TERT promoter mutations-based tumor DNA detection in patients with bladder cancer: A pilot study.

Telomerase reverse transcriptase (TERT) promoter mutations are the most frequent genetic events in bladder cancer (BC). The aim of the present pilot study was to evaluate the diagnostic potential of urine TERT promoter mutations-based liquid biopsy in patients with an ongoing oncological process, as well as in post-resection patients at risk of BC recurrence. A total of 60 patients were enrolled, of whom 27 patients had histologically proven BC; 23 had no signs of BC (control group); and 10 patients underwent transurethral malignancy resection 3-6 months prior to urine donation ('second look' group). Urine TERT promoter mutations were detected using Droplet Digital PCR. Receiver operating characteristic curve analysis revealed significant diagnostic power of the present approach (area under the curve: -0.768). At the cut-off value of tumor DNA fraction 0.34%, the sensitivity and specificity were 55.56 and 100%, respectively. In the positive samples, tumor DNA fraction varied significantly from 0.59 to 48.77%. In the 'second look' group, tumor DNA was detected in 4/10 patients, highlighting the possibility of BC recurrence with its fraction ranging only from 0.90 to 6.61%. Therefore, urine TERT promoter mutations-based liquid biopsy appears to be a promising tool for BC diagnosis and surveillance. The main study will include recruitment of additional patients, extension of the mutation panel, prolonged follow-up of the post-resection patients, as well as screening of industrial workers exposed to specific carcinogens.

Diaminorhodamine and Light-Activatable NO Donors: Photorelease Quantification and Potential Pitfalls.

Light-activatable nitric oxide (NO) donors have become of interest in the recent years. They produce NO when illuminated by light, which enables the control of its local concentration and is promising for biomedical applications. Several successful prototypes of photodonors have been published, but further research is needed to improve their properties such as water-solubility, activation wavelength, biocompatibility etc. One of major challenges on this way is to evaluate the efficiency of NO generation. Several methods may be used to track NO, including spin traps, specific electrodes and fluorescence-based probes. We have studied the applicability of well-known fluorescent reporter, diaminorhodamine (DAR-2), for the evaluation of NO production by photodonors. Our results indicate that DAR-2 can be used for the quantification of NO photorelease if this process is not accompanied by the singlet oxygen formation. Otherwise the oxidation of probe results in huge fluorescence increase, which interferes with signal due to reaction with NO. This issue should be taken into account when studying hybrids releasing both NO and 1O2, which are promising for photodynamic therapy.

Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer.

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

Activating Telomerase TERT Promoter Mutations and Their Application for the Detection of Bladder Cancer.

This review summarizes state-of-the-art knowledge in early-generation and novel urine biomarkers targeting the telomerase pathway for the detection and follow-up of bladder cancer (BC). The limitations of the assays detecting telomerase reactivation are discussed and the potential of transcription-activating mutations in the promoter of the TERT gene detected in the urine as promising simple non-invasive BC biomarkers is highlighted. Studies have shown good sensitivity and specificity of the urinary TERT promoter mutations in case-control studies and, more recently, in a pilot prospective cohort study, where the marker was detected up to 10 years prior to clinical diagnosis. However, large prospective cohort studies and intervention studies are required to fully validate their robustness and assess their clinical utility. Furthermore, it may be interesting to evaluate whether the clinical performance of urinary TERT promoter mutations could increase when combined with other simple urinary biomarkers. Finally, different approaches for assessment of TERT promoter mutations in urine samples are presented together with technical challenges, thus highlighting the need of careful technological validation and standardization of laboratory methods prior to translation into clinical practice.