Abstract
Somatic mutations in the telomerase reverse transcriptase (TERT) promoter region are highly frequent in urothelial cancer (UC), and their detection in urine (cell-free DNA from the urine supernatant or DNA from exfoliated cells in the urine pellet) has demonstrated promising evidence as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumour-derived mutations in urine requires highly sensitive methods, capable of measuring low-allelic fraction mutations. We developed sensitive droplet digital PCR (ddPCR) assays for detecting urinary TERT promoter mutations (uTERTpm), targeting the two most common mutations (C228T and C250T), as well as the rare A161C, C228A, and CC242-243TT mutations. Here, we described the step-by-step protocol uTERTpm mutation screening using simplex ddPCR assays and give some recommendations for isolation of DNA from urine samples. We also provide limits of detection for the two most frequent mutations and discuss advantages of the method for clinical implementation of the assays for the detection and monitoring of UC.
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Acknowledgements
The work was supported by the French Cancer League, the Russian Foundation for Basic Research and the International Agency for Research on Cancer.
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Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this chapter, and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.
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Zvereva, M. et al. (2023). Simplex Droplet Digital PCR Assays for the Detection of TERT Promoter Mutations in Urine Samples for the Non-invasive Diagnosis of Urothelial Cancer. In: Hoffmann, M.J., Gaisa, N.T., Nawroth, R., Ecke, T.H. (eds) Urothelial Carcinoma. Methods in Molecular Biology, vol 2684. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3291-8_13
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DOI: https://doi.org/10.1007/978-1-0716-3291-8_13
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