Do Hongdo, Molania Ramyar, Mitchell Paul L, Vaiskunaite Rita, Murdoch John D, Dobrovic Alexander
Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Australia;
School of Cancer Medicine, La Trobe University, Melbourne, Australia.
Clin Chem. 2017 Sep;63(9):1506-1514. doi: 10.1373/clinchem.2017.271932. Epub 2017 Jul 18.
False-positive T790M mutations have been reported in formalin-fixed lung tumors, but the cause of the false positives has not been identified. The T790M mutation results from a C>T change at the cytosine of a CpG dinucleotide. The presence or absence of methylation at this cytosine has different consequences following deamination, resulting in a thymine or uracil, respectively, both of which however result in an artifactual change. Uracil-DNA glycosylase (UDG) can be used to eliminate DNA templates with uracil residues but is not active against artifactual thymines. We therefore investigated the use of thymine-DNA glycosylase (TDG) to reduce artifactual T790M mutations.
Formalin-fixed normal lung tissues and lung squamous cell carcinomas were tested to measure the frequency of false-positive mutations by use of droplet digital PCR before and after treatment with either UDG or TDG. Methylation at the cytosine at T790 was assessed by pyrosequencing and by analysis of public databases.
Artifactual T790M mutations were detected in all of the archival formalin-fixed normal lung and lung squamous cell carcinomas at mutant allele frequencies of 1% or lower. The cytosine at T790 showed high levels of methylation in all lung cancer samples and normal tissues. Pretreatment of the formalin-fixed DNA with either UDG or TDG reduced the false T790M mutations, but a greater reduction was seen with the TDG treatment.
Both U:G and T:G lesions in formalin-fixed tissue are sources of false-positive T790M mutations. This is the first report of the use of TDG to reduce sequence artifacts in formalin-fixed DNA and is applicable to the accurate detection of mutations arising at methylated cytosines.
在福尔马林固定的肺肿瘤中已报道存在T790M假阳性突变，但假阳性的原因尚未明确。T790M突变是由CpG二核苷酸中胞嘧啶的C>T变化引起的。该胞嘧啶甲基化与否在脱氨后会产生不同后果，分别导致胸腺嘧啶或尿嘧啶，然而二者都会导致人为变化。尿嘧啶-DNA糖基化酶（UDG）可用于消除带有尿嘧啶残基的DNA模板，但对人为产生的胸腺嘧啶无活性。因此，我们研究了使用胸腺嘧啶-DNA糖基化酶（TDG）来减少人为的T790M突变。
对福尔马林固定的正常肺组织和肺鳞状细胞癌进行检测，在使用UDG或TDG处理前后，通过液滴数字PCR测量假阳性突变的频率。通过焦磷酸测序和分析公共数据库评估T790位点胞嘧啶的甲基化情况。
在所有存档的福尔马林固定的正常肺组织和肺鳞状细胞癌中均检测到人为的T790M突变，突变等位基因频率为1%或更低。T790位点的胞嘧啶在所有肺癌样本和正常组织中均显示出高水平的甲基化。用UDG或TDG对福尔马林固定的DNA进行预处理可减少假T790M突变，但TDG处理的减少幅度更大。
福尔马林固定组织中的U:G和T:G损伤都是T790M假阳性突变的来源。这是关于使用TDG减少福尔马林固定DNA序列假象的首次报道，适用于准确检测甲基化胞嘧啶处产生的突变。
