Ward Douglas G, Baxter Laura, Gordon Naheema S, Ott Sascha, Savage Richard S, Beggs Andrew D, James Jonathan D, Lickiss Jennifer, Green Shaun, Wallis Yvonne, Wei Wenbin, James Nicholas D, Zeegers Maurice P, Cheng K K, Mathews Glenn M, Patel Prashant, Griffiths Michael, Bryan Richard T
Institute of Cancer & Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom.
Warwick Systems Biology Centre, University of Warwick, Coventry, CV4 7AL, United Kingdom.
PLoS One. 2016 Feb 22;11(2):e0149756. doi: 10.1371/journal.pone.0149756. eCollection 2016.
Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.
DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).
Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).
This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.
迄今为止，用于检测原发性或复发性膀胱癌的高灵敏度和特异性尿液检测方法仍难以实现。我们对膀胱癌基因组畸变的了解不断增加，这应该能够推动基于尿液DNA的此类检测方法的开发。
从尿细胞沉淀中提取DNA，并使用聚合酶链反应（PCR）扩增端粒酶逆转录酶（TERT）启动子区域以及成纤维细胞生长因子受体3（FGFR3）、磷脂酰肌醇-3激酶催化亚基α（PIK3CA）、肿瘤蛋白p53（TP53）、哈维鼠肉瘤病毒癌基因同源物（HRAS）、赖氨酸特异性去甲基化酶6A（KDM6A）和维甲酸受体α（RXRA）的编码区域，这些区域在膀胱癌中经常发生突变。对PCR产物进行条形码标记、混合，并在Illumina MiSeq上进行双端2×250bp测序。分析了20名非癌症对照、120名原发性膀胱癌患者（41例pTa期、40例pT1期、39例pT2 +期）和91例经尿道膀胱肿瘤电切术（TURBT）后的膀胱癌患者（89例无癌）的尿液DNA。
尽管从一些尿细胞沉淀中提取的DNA量很少，但96%的样本产生的平均读取深度>500。仅分析先前报道的点突变，在55%的膀胱癌患者中发现TERT突变（与分期无关），30%的膀胱癌患者中发现FGFR3突变，14%的膀胱癌患者中发现PIK3CA突变，12%的膀胱癌患者中发现TP53突变。总体而言，在122例膀胱癌患者中的86例中检测到了这些先前报道的膀胱癌突变（灵敏度为70%），而在109例未检测到膀胱癌的患者中仅3例检测到（特异性为97%）。
这种简单、经济高效的方法可用于对携带这些突变的非肌层浸润性膀胱癌患者进行无创监测。该方法对DNA输入要求低，能够在大量正常DNA中检测到低水平的突变DNA。这些基因代表了一个最小的生物标志物组合，可在此基础上添加额外的标志物，以开发一种高灵敏度的膀胱癌诊断检测方法。
