Zvereva Maria, Hosen Md Ismail, Forey Nathalie, Sheikh Mahdi, Kannengiesser Caroline, Ba Ibrahima, Manel Arnaud, Vian Emmanuel, Le Calvez-Kelm Florence
Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
Genomic Epidemiology Branch, International Agency for Research on Cancer (IARC), Lyon, France.
Methods Mol Biol. 2023;2684:213-228. doi: 10.1007/978-1-0716-3291-8_13.
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.
端粒酶逆转录酶（TERT）启动子区域的体细胞突变在尿路上皮癌（UC）中非常常见，在尿液中检测这些突变（尿液上清液中的游离DNA或尿沉渣中脱落细胞的DNA）已显示出有望作为UC检测和监测的非侵入性生物标志物的证据。然而，在尿液中检测这些肿瘤衍生的突变需要高度灵敏的方法，能够检测低等位基因分数的突变。我们开发了灵敏的液滴数字PCR（ddPCR）检测方法，用于检测尿TERT启动子突变（uTERTpm），针对两种最常见的突变（C228T和C250T）以及罕见的A161C、C228A和CC242 - 243TT突变。在此，我们描述了使用单重ddPCR检测方法进行uTERTpm突变筛查的分步方案，并给出了从尿液样本中分离DNA的一些建议。我们还提供了两种最常见突变的检测限，并讨论了该方法在临床实施UC检测和监测检测中的优势。
