Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.
Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.
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DOI：
10.1158/1078-0432.ccr-17-0291
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发表时间：
2017-09-15
期刊：
影响因子：
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通讯作者：
Salathia NS
中科院分区：
文献类型：
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作者：
Janku F;Zhang S;Waters J;Liu L;Huang HJ;Subbiah V;Hong DS;Karp DD;Fu S;Cai X;Ramzanali NM;Madwani K;Cabrilo G;Andrews DL;Zhao Y;Javle M;Kopetz ES;Luthra R;Kim HJ;Gnerre S;Satya RV;Chuang HY;Kruglyak KM;Toung J;Zhao C;Shen R;Heymach JV;Meric-Bernstam F;Mills GB;Fan JB;Salathia NS
血浆中肿瘤来源的无细胞DNA （cfDNA）可用于分子检测，为肿瘤组织提供了一种有吸引力的替代方法。常用的基于pcr的技术一次只能检测有限数量的变异。因此，需要能够检测广谱分子变化的新型超灵敏技术来进一步个性化癌症治疗。我们开发了一种高灵敏度的超深下一代测序（NGS）检测方法，使用TruSeq纳米文库制备试剂和Nextera Rapid Capture目标富集试剂盒，生成血浆cfDNA测序文库，用于61种癌症相关基因的突变分析，使用常见的生物信息学工具。回顾性比较了在不同临床护理点获得的档案原发性或转移性肿瘤组织的分子检测结果。在一项55例晚期癌症患者的研究中，超深NGS检测检测出82%（完全检测）至87%（完全和部分检测）在不一致收集的相应档案肿瘤组织中鉴定出的畸变。突变cfDNA变异等位基因频率（VAF）低的患者比VAF高的患者存活时间更长（P=0.018）。在接受全身治疗的患者中，放射学反应与cfDNA VAF的变化呈正相关（P=0.02），与突变cfDNA VAF不变/升高相比，cfDNA VAF降低与治疗失败时间延长相关（TTF； P=0.03）。与传统的临床档案标本突变检测相比，超深NGS检测具有良好的敏感性。突变cfDNA的高VAF与较短的生存期相对应。突变cfDNA的VAF变化与TTF相关。
Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. We developed a highly sensitive ultra-deep next-generation sequencing (NGS) assay using reagents from TruSeq Nano library preparation and Nextera Rapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared to molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. In a study of 55 patients with advanced cancer, the ultra-deep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P=0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P=0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P=0.03). Ultra-deep NGS assay has good sensitivity compared to conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF.