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首页> 外文期刊>The Journal of molecular diagnostics: JMD >Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.
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Detection of KRAS and BRAF mutations in colorectal carcinoma roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping.

机译：检测KRAS和BRAF突变在大肠癌中的作用，用于高灵敏度锁定核酸PCR测序和广谱质谱基因分型。

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摘要

KRAS and BRAF mutations predict the resistance of colorectal carcinomas to therapy targeted to the epidermal growth factor receptor, but their detection can be challenging because of high testing volume, frequently low tumor content, and the spectrum of rarer mutations in these genes. To address these issues, we evaluated a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant DNA, and we also evaluated a mass spectrometry genotyping assay (Sequenom, San Diego, CA) that is suitable for broad mutation screening. Clinical cases (n = 308) previously tested for KRAS and BRAF by standard sequencing were retested by LNA-PCR sequencing incorporating an LNA oligonucleotide to suppress amplification of nonmutant DNA, and by a Sequenom assay panel targeting common mutations in both genes. Standard sequencing detected 121 KRAS (39%) and 10 BRAF mutations; retesting with the LNA-based method and the Sequenom assay detected 19 (140/308, 45%) and 6 (127/308, 41%) additional KRAS mutants, respectively. One additional BRAF mutant was detected by the Sequenom assay. The analytical sensitivities were 0.3% for both KRAS and BRAF by LNA-PCR and from 1% to 10% for the Sequenom assays, depending on the specific mutation. Given these results, standard sequencing is suboptimal for mutation detection in metastatic and treated lesions even with predissection for tumor enrichment. High-sensitivity LNA-PCR sequencing detects significantly more mutations, whereas the Sequenom platform shows intermediate sensitivity but offers significant advantages for broader mutation screening.
机译：KRAS和BRAF突变预测了结直肠癌对靶向表皮生长因子受体的疗法的耐药性，但由于检测量大，肿瘤含量低和这些基因中罕见突变的范围广，它们的检测可能具有挑战性。为了解决这些问题，我们评估了锁定核酸（LNA）-PCR测序测定法以检测低水平的突变体DNA，还评估了适用于广泛突变筛选的质谱基因分型测定法（Sequenom，圣地亚哥，加利福尼亚） 。以前通过标准测序测试过KRAS和BRAF的临床病例（n = 308）通过结合LNA寡核苷酸以抑制非突变DNA扩增的LNA-PCR测序和针对这两个基因共同突变的Sequenom分析小组进行了重新测试。标准测序检测到121个KRAS（39％）和10个BRAF突变;使用基于LNA的方法进行重新测试和Sequenom分析分别检测到19个（140/308，45％）和6个（127/308，41％）额外的KRAS突变体。通过Sequenom分析检测到另外一种BRAF突变体。通过LNA-PCR，对KRAS和BRAF的分析灵敏度分别为0.3％，对Sequenom分析的灵敏度为1％至10％，具体取决于特定的突变。鉴于这些结果，即使对转移灶和已治疗的病变进行了肿瘤富集，标准测序对于突变检测的次优选择也不理想。高灵敏度LNA-PCR测序可检测到明显更多的突变，而Sequenom平台显示出中等灵敏度，但为更广泛的突变筛选提供了显着优势。

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