使用全自动平台中的直接DNA移液和下一代测序技术优化实验室工作流程，评估转移性结直肠癌患者的KRAS，NRAS和BRAF热点突变检测。,PLOS ONE

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使用全自动平台中的直接DNA移液和下一代测序技术优化实验室工作流程，评估转移性结直肠癌患者的KRAS，NRAS和BRAF热点突变检测。
PLOS ONE ( IF 2.9 ) Pub Date : 2019-07-02 , DOI: 10.1371/journal.pone.0219204
Pauline Gilson _{1,

2} , Claire Franczak ₂ , Ludovic Dubouis ₂ , Marie Husson ₂ , Marie Rouyer ₂ , Jessica Demange ₂ , Marie Perceau ₂ , Agnès Leroux ₂ , Jean-Louis Merlin _{1,

2} , Alexandre Harlé _{1,

2}

Affiliation

背景技术对KRAS，NRAS（RAS）和BRAF突变的评估是转移性大肠癌（mCRC）患者管理的标准。可以使用下一代测序（NGS）或基于实时PCR的测定法评估突变。对于NGS，获得结果的时间为1-2周，对于基于PCR的实时测定，则为1-3天。使用NGS可能会延迟一线治疗的开始，而使用基于PCR的测定法则受到可能分析靶标数量的限制。Idylla系统是一种基于实时PCR盒的检测方法，能够使用一部分FFPE肿瘤组织样品分析热点突变。为了结合短暂的延迟和对大型基因面板的分析，我们在这里提出了一个结合Idylla系统和NGS的实验室工作流程，并与组织量少的FFPE样品兼容。在这项研究中，我们通过直接吸取盒中的DNA代替制造商推荐的FFPE切片中的DNA来评估和验证Idylla系统，以分析RAS和BRAF突变。材料与方法用Idylla KRAS和Idylla NRAS-BRAF突变测试对从具有NGS特征性RAS和BRAF突变的mCRC患者的29份FFPE样品中提取的DNA进行检测，以评估每种测试的敏感性，特异性，可重复性和检测极限。结果在NGS与Idylla结果之间发现100％的一致性，用于确定KRAS（12/12），NRAS（12/12）和BRAF（11/11）突变，敏感性和特异性为100％。该系统显示出良好的重现性，CV低于3％。用KRAS和NRAS突变的2.5 ng DNA和BRAF突变的5 ng DNA达到LOD。

"点击查看英文标题和摘要"

Evaluation of KRAS, NRAS and BRAF hotspot mutations detection for patients with metastatic colorectal cancer using direct DNA pipetting in a fully-automated platform and Next-Generation Sequencing for laboratory workflow optimisation.

BACKGROUND Assessment of KRAS, NRAS (RAS) and BRAF mutations is a standard in the management of patients with metastatic colorectal cancer (mCRC). Mutations could be assessed using next-generation sequencing (NGS) or real-time PCR-based assays. Times to results are 1 to 2 weeks for NGS and 1 to 3 days for real-time PCR-based assays. Using NGS can delay first-line treatment commencement and using PCR-based assays is limited by the number of possible analysed targets. The Idylla system is a real-time PCR cartridge-based assay, able to analyse hotspots mutations using one section of FFPE tumour tissue sample. To combine short delays and analysis of a large gene-panel, we propose here a laboratory workflow combining the Idylla system and NGS and compatible with FFPE samples with low tissue quantity. In this study we evaluated and validated the Idylla system for the analysis of RAS and BRAF mutations by pipetting directly DNA in the cartridge instead of FFPE section as recommended by the manufacturer. MATERIALS AND METHODS DNA extracted from 29 FFPE samples from mCRC patients with NGS-characterized RAS and BRAF mutations were tested with the Idylla KRAS and the Idylla NRAS-BRAF mutation tests to assess sensitivity, specificity, reproducibility and limit of detection of each test. RESULTS A 100% concordance was found between NGS and Idylla results for the determination of KRAS (12/12), NRAS (12/12) and BRAF (11/11) mutations with a sensitivity and a specificity of 100%. The system showed a good reproducibility with CV inferior to 3%. LOD was reached with 2.5 ng of DNA for KRAS and NRAS mutations and 5 ng of DNA for BRAF mutations. CONCLUSIONS The analysis of RAS and BRAF mutations using DNA pipetted directly in the cartridge of the Idylla system showed a good sensitivity, specificity, reproducibility and LOD, and can be integrated in a laboratory workflow for samples with few tissue without compromising a further complete tumour characterization using NGS.

更新日期：2019-07-03

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