Detection of somatic mutations in tumor tissues helps to understand tumor biology and guide treatment selection. Methods such as quantitative PCR can analyze a few mutations with high efficiency, while next generation sequencing (NGS) based methods can analyze hundreds to thousands of mutations. However, there is a lack of cost-effective method for quantitatively analyzing tens to a few hundred mutations of potential biological and clinical significance. Through a comprehensive database and literature review we selected 299 mutations associated with colorectal cancer. We then designed a highly multiplexed assay panel (8-wells covering 299 mutations in 109 genes) based on an automated MADLI-TOF mass spectrometry (MS) platform. The multiplex panel was tested with a total of 319 freshly frozen tissues and 92 FFPE samples from 229 colorectal cancer patients, with 13 samples also analyzed by a targeted NGS method covering 532 genes. Multiplex somatic mutation panel based on MALDI-TOF MS detected and quantified at least one somatic mutation in 142 patients, with KRAS, TP53 and APC being the most frequently mutated genes. Extensive validation by both capillary sequencing and targeted NGS demonstrated high accuracy of the multiplex MS assay. Out of 35 mutations tested with plasmid constructs, sensitivities of 5 and 10% mutant allele frequency were achieved for 19 and 16 mutations, respectively. Automated MALDI-TOF MS offers an efficient and cost-effective platform for highly multiplexed quantitation of 299 somatic mutations, which may be useful in studying the biological and clinical significance of somatic mutations with large numbers of cancer tissues.

中文翻译：

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通过自动 MALDI-TOF 质谱法对结直肠癌患者的 299 种体细胞突变进行高度多重定量


检测肿瘤组织中的体细胞突变有助于了解肿瘤生物学并指导治疗选择。定量PCR等方法可以高效分析少数突变，而基于下一代测序（NGS）的方法可以分析数百到数千个突变。然而，缺乏经济有效的方法来定量分析数十到数百个具有潜在生物学和临床意义的突变。通过综合数据库和文献综述，我们选择了 299 个与结直肠癌相关的突变。然后，我们基于自动化 MADLI-TOF 质谱 (MS) 平台设计了一个高度多重检测面板（8 孔，涵盖 109 个基因的 299 个突变）。该多重检测组使用了来自 229 名结直肠癌患者的总共 319 个新鲜冷冻组织和 92 个 FFPE 样本进行了测试，其中 13 个样本还通过涵盖 532 个基因的靶向 NGS 方法进行了分析。基于 MALDI-TOF MS 的多重体细胞突变面板检测并量化了 142 名患者中的至少一种体细胞突变，其中 KRAS、TP53 和 APC 是最常见的突变基因。通过毛细管测序和靶向 NGS 进行的广泛验证证明了多重 MS 检测的高精度。在用质粒构建体测试的 35 个突变中，19 个和 16 个突变分别达到了 5% 和 10% 突变等位基因频率的灵敏度。自动化 MALDI-TOF MS 为 299 种体细胞突变的高度多重定量提供了一个高效且经济高效的平台，这可能有助于研究大量癌症组织中体细胞突变的生物学和临床意义。