Ivanov Maxim, Laktionov Konstantin, Breder Valery, Chernenko Polina, Novikova Ekaterina, Telysheva Ekaterina, Musienko Sergey, Baranova Ancha, Mileyko Vladislav
Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow Region, 141700, Russia.
Atlas Biomed Group, Moscow, 121069, Russia.
J Transl Med. 2017 Jan 31;15(1):22. doi: 10.1186/s12967-017-1125-8.
Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved.
DNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run. Sequencing data were comparatively analyzed by several bioinformatics pipelines.
Libraries yielded sufficient coverage to detect even low prevalent mutations. We found that the number of FFPE sequence artifacts significantly correlates with pre-normalization concentration of libraries (rank correlation -0.81; p < 1e-10), thus, contributing to sample-specific variant detection cut-offs. Surprisingly, extensive validation of EGFR mutation calls by a combination of aligners and variant callers resulted in identification of two false negatives and one false positive that were due to complexity of underlying genomic change, confirmed by Sanger sequencing. Additionally, the study of the non-EGFR amplicons revealed 33 confirmed unique mutations in 17 genes, with TP53 being the most frequently mutated. Clinical relevance of these finding is discussed.
Reporting of entire mutational spectrum revealed by targeted sequencing is questionable, at least until the clinically-driven guidelines on reporting of somatic mutations are established. The standardization of sequencing protocols, especially their data analysis components, requires assay-, disease-, and, in many cases, even sample-specific customization that could be performed only in cooperation with clinicians.
在精准肿瘤学框架内，下一代测序技术有潜力彻底改变癌症患者的管理方式。然而，缺乏标准化减缓了该技术进入临床检测领域的进程。在此，我们剖析了肿瘤学中NGS诊断的一些常见问题，并介绍了可解决这些问题的方法。
从26个福尔马林固定石蜡包埋（FFPE）标本中提取DNA，并用靶向48个癌症相关基因的TrueSeq Amplicon Cancer Panel（Illumina公司，加利福尼亚州圣地亚哥）进行处理，然后单次运行测序。测序数据通过几种生物信息学流程进行比较分析。
文库产生了足够的覆盖度，甚至能够检测到低频率的突变。我们发现FFPE序列伪像的数量与文库归一化前的浓度显著相关（等级相关系数为 -0.81；p < 1e-10），因此有助于确定样本特异性变异检测阈值。令人惊讶的是，通过比对器和变异调用器组合对EGFR突变进行广泛验证时，发现了两例假阴性和一例假阳性结果，这是由于潜在基因组变化的复杂性导致的，经Sanger测序证实。此外，对非EGFR扩增子的研究在17个基因中发现了33个经确认的独特突变，其中TP53是突变最频繁的基因。讨论了这些发现的临床相关性。
至少在建立由临床驱动的体细胞突变报告指南之前，报告靶向测序揭示的整个突变谱是有问题的。测序方案的标准化，尤其是其数据分析部分，需要根据检测方法、疾病，在许多情况下甚至需要根据样本进行定制，这只能与临床医生合作完成。
