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.

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TERT Promoter Mutation Analysis of Whole-Organ Mapping Bladder Cancers

Genes ◽

10.3390/genes12020230 ◽

2021 ◽

Vol 12 (2) ◽

pp. 230

Author(s):

Veronika Weyerer ◽

Markus Eckstein ◽

Pamela L. Strissel ◽

Adrian Wullweber ◽

Fabienne Lange ◽

...

Keyword(s):

Bladder Cancer ◽

Telomere Length ◽

Hot Spot ◽

Tumor Development ◽

Non Invasive ◽

Tert Promoter ◽

Mutational Status ◽

Tert Promoter Mutations ◽

Promoter Mutations ◽

Normal Urothelium

Background: Multifocal occurrence is a main characteristic of urothelial bladder cancer (UBC). Whether urothelial transformation is caused by monoclonal events within the urothelium, or by polyclonal unrelated events resulting in several tumor clones is still under debate. TERT promoter mutations are the most common somatic alteration identified in UBC. In this study, we analyzed different histological tissues from whole-organ mapping bladder cancer specimens to reveal TERT mutational status, as well as to discern how tumors develop. Methods: Up to 23 tissues from nine whole-organ mapping bladder tumor specimens, were tested for TERT promoter mutations including tumor associated normal urothelium, non-invasive urothelial lesions (hyperplasia, dysplasia, metaplasia), carcinoma in situ (CIS) and different areas of muscle invasive bladder cancers (MIBC). The mutational DNA hotspot region within the TERT promoter was analyzed by SNaPshot analysis including three hot spot regions (−57, −124 or −146). Telomere length was measured by the Relative Human Telomere Length Quantification qPCR Assay Kit. Results: TERT promoter mutations were identified in tumor associated normal urothelium as well as non-invasive urothelial lesions, CIS and MIBC. Analysis of separate regions of the MIBC showed 100% concordance of TERT promoter mutations within a respective whole-organ bladder specimen. Polyclonal events were observed in five out of nine whole-organ mapping bladder cancers housing tumor associated normal urothelium, non-invasive urothelial lesions and CIS where different TERT promoter mutations were found compared to MIBC. The remaining four whole-organ mapping bladders were monoclonal for TERT mutations. No significant differences of telomere length were observed. Conclusions: Examining multiple whole-organ mapping bladders we conclude that TERT promoter mutations may be an early step in bladder cancer carcinogenesis as supported by TERT mutations detected in tumor associated normal urothelium as well as non-invasive urothelial lesions. Since mutated TERT promoter regions within non-invasive urothelial lesions are not sufficient alone for the establishment of cancerous growth, this points to the contribution of other gene mutations as a requirement for tumor development.

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Non-invasive prediction of recurrence in bladder cancer by detecting somatic TERT promoter mutations in urine

British Journal of Cancer ◽

10.1038/bjc.2017.210 ◽

2017 ◽

Vol 117 (4) ◽

pp. 583-587 ◽

Cited By ~ 34

Author(s):

Françoise Descotes ◽

Norelyakin Kara ◽

Myriam Decaussin-Petrucci ◽

Eric Piaton ◽

Florence Geiguer ◽

...

Keyword(s):

Bladder Cancer ◽

Non Invasive ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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Non invasive prediction of recurrences in bladder cancer by detecting TERT promoter mutations in urine

European Urology Supplements ◽

10.1016/s1569-9056(17)30186-0 ◽

2017 ◽

Vol 16 (3) ◽

pp. e195-e197

Author(s):

N. Kara ◽

F. Descotes ◽

M. Decaussin Petrucci ◽

E. Piaton ◽

F. Geiguer ◽

...

Keyword(s):

Bladder Cancer ◽

Non Invasive ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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The role of telomerase reverse transcriptase (TERT) promoter mutations in prognosis in bladder cancer

Bioengineered ◽

10.1080/21655979.2021.1915725 ◽

2021 ◽

Vol 12 (1) ◽

pp. 1495-1504

Author(s):

Song Wan ◽

Xuan Liu ◽

Wei Hua ◽

Ming Xi ◽

Yulin Zhou ◽

...

Keyword(s):

Bladder Cancer ◽

Reverse Transcriptase ◽

Telomerase Reverse Transcriptase ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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Urinary TERT promoter mutations are detectable up to 10 years prior to clinical diagnosis of bladder cancer: Evidence from the Golestan Cohort Study

EBioMedicine ◽

10.1016/j.ebiom.2020.102643 ◽

2020 ◽

Vol 53 ◽

pp. 102643 ◽

Cited By ~ 5

Author(s):

Md Ismail Hosen ◽

Mahdi Sheikh ◽

Maria Zvereva ◽

Ghislaine Scelo ◽

Nathalie Forey ◽

...

Keyword(s):

Bladder Cancer ◽

Cohort Study ◽

Clinical Diagnosis ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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Occurrence, functionality, and abundance of the TERT promoter mutations

10.1101/2021.05.03.442397 ◽

2021 ◽

Author(s):

Sivaramakrishna Rachakonda ◽

Joerg D. Hoheisel ◽

Rajiv Kumar

Keyword(s):

Replicative Senescence ◽

Replication Process ◽

Telomere Shortening ◽

Tert Promoter ◽

Oncogenic Pathways ◽

Whole Genomes ◽

Tert Gene ◽

Tert Promoter Mutations ◽

Promoter Mutations ◽

Pan Cancer

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 rejuvenation of telomerase via different mechanisms. Mutations within the promoter of the telomerase reverse transcriptase (TERT) gene define a definite method for the ribonucleic enzyme regeneration predominantly in cancers that arise from tissues with low rates of self-renewal. The promoter mutations cause a moderate surge in TERT transcription and telomerase rejuvenation 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 and publications have resolved the discrete aspects, effects, and clinical relevance of the TERT promoter mutations. Those noncoding alterations link the TERT transcription 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 and the evolutionary constraints limit the functional fraction within the human genome, hence the lack of abundant mutations outside the coding sequences.

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

Cancers ◽

10.3390/cancers12123541 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3541 ◽

Cited By ~ 2

Author(s):

Md Ismail Hosen ◽

Nathalie Forey ◽

Geoffroy Durand ◽

Catherine Voegele ◽

Selin Bilici ◽

...

Keyword(s):

Urothelial Cancer ◽

Digital Pcr ◽

Kappa Coefficient ◽

Droplet Digital Pcr ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

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.

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Cell-free tumor DNA and TERT promoter mutations in bladder cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.353 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. 353-353 ◽

Cited By ~ 1

Author(s):

Franklin W. Huang ◽

Mikael L. Rinne ◽

Kevin T. Lundgren ◽

Stephanie Anne Mullane ◽

Irene Moreno ◽

...

Keyword(s):

Urothelial Carcinoma ◽

Metastatic Disease ◽

Tert Promoter Mutation ◽

Plasma Samples ◽

Promoter Mutation ◽

Library Construction ◽

Non Invasive ◽

Tert Promoter ◽

Tert Promoter Mutations ◽

Promoter Mutations

353 Background: Currently, there are no FDA-approved blood biomarkers for the prognosis or prediction of outcomes in urothelial carcinoma (UC). The telomerase reverse transcriptase ( TERT) promoter is recurrently mutated at high frequency in UC (50%). These mutations have been correlated with tumor recurrence and survival. Tumor cell-free DNA (cfDNA) with somatic genomic alterations can be found in the plasma of cancer patients and has the potential for use as a non-invasive cancer biomarker. Detection of TERT promoter mutations in cfDNA might be used as a prognostic tool to monitor disease outcome in UC patients. We set out to detect tumor cfDNA and TERT promoter mutations in cfDNA from patients with UC at different stages. Methods: UC patients receiving chemotherapy in the neoadjuvant, first or second-line metastatic setting had blood collected either before or during therapy. cfDNA was isolated from ~1ml plasma samples using the QIAmp (Qiagen) kit. Samples underwent ultra-low pass whole genome sequencing (ULP-WGS) to determine whether tumor cfDNA could be detected in these samples. TERT promoter mutations were detected using a sensitive qPCR assay. Results: 40 plasma samples from a total of 32 patients with urothelial carcinoma were analyzed. Sufficient amounts of plasma cfDNA were obtained for library construction and ULP-WGS in 11 patients. 6 of these 11 patients were determined to be positive for detectable tumor cfDNA and of these, all were metastatic and 50% (3/6) were positive for a TERT promoter mutation. In total, 8 out of 40 samples (20%) were positive for a TERT promoter mutation, including samples from two patients where total cfDNA yield was insufficient for library construction. A total of ~20% of patients with metastatic disease were positive for TERT promoter mutations in cfDNA. The low percentage of samples having sufficient cfDNA most likely reflects the low volume of plasma used. Conclusions: TERT promoter mutations were identified in cfDNA of UC patients. ULP-WGS showed tumor cfDNA in patients with a high tumor burden and metastatic disease. TERTpromoter mutations in cfDNA could potentially be used as a non-invasive method for detection of disease. These results have implications for the use of cfDNA in the evaluation of advanced UC.

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