Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients.
Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients.
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DOI：
10.1038/bjc.2017.8
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发表时间：
2017-03-14
影响因子：
8.8
通讯作者：
Troncone G
中科院分区：
文献类型：
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作者：
Malapelle U;Mayo de-Las-Casas C;Rocco D;Garzon M;Pisapia P;Jordana-Ariza N;Russo M;Sgariglia R;De Luca C;Pepe F;Martinez-Bueno A;Morales-Espinosa D;González-Cao M;Karachaliou N;Viteri Ramirez S;Bellevicine C;Molina-Vila MA;Rosell R;Troncone G
当肿瘤组织不可用时，无细胞DNA（cfDNA）可以作为遗传分析的替代品。由于cfDNA中突变的等位基因通常低于1%，因此下一代测序（NGS）必须缩小范围，仅靶向临床相关基因。在这项概念验证研究中，我们开发了一个用于超深度测序的面板，以识别cfDNA中的此类突变。我们的小组（“SiRe”）涵盖了涉及非小细胞肺癌（NSCLC）、胃肠道间质瘤、结直肠癌和黑色素瘤的6个基因（EGFR、KRAS、NRAS、BRAF、cKIT和PDGFRα）中的568个突变。我们分三步评估了面板性能。首先，我们分析了其对细胞系DNA的分析灵敏度，并通过使用在不同基因中具有多个突变的人工参考标准。其次，我们分析了来自癌症患者的cfDNA，包括就诊（n=42）、治疗反应（n=12）和肿瘤进展（n=11）;所有患者都有配对的肿瘤组织和cfDNA，之前用Taqman衍生测定法（TDA）进行了基因分型。第三，我们测试了从NSCLC患者（n=79）前瞻性收集的血液样本，以评估SiRe在临床实践中的性能。SiRe具有较高的分析性能和0.01%的检测下限。在回顾性系列中，SiRe检测到40个EGFR、11个KRAS、1个NRAS和5个BRAF突变（与TDA的一致性为96.8%）。在基线样本中，SiRe相对于肿瘤组织具有100%的特异性和79%的灵敏度。最后，在前瞻性系列中，SiRe在基线时检测到8.7%（4/46）的EGFR突变，在肿瘤进展患者中检测到42.9%（9/21）的EGFR p.T790M突变。SiRe是用于临床实践中cfDNA分析的可行的NGS组。
When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. Our panel (‘SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.