Université de Lorraine, CNRS UMR 7039 CRAN, Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandœuvre-lès-Nancy, France.
Institut de Cancérologie de Lorraine, Service de Biopathologie, Vandœuvre-lès-Nancy, France.
PLoS One. 2019 Jul 2;14(7):e0219204. doi: 10.1371/journal.pone.0219204. eCollection 2019.
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.
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.
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.
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.
KRAS、NRAS（RAS）和 BRAF 突变的评估是转移性结直肠癌（mCRC）患者管理的标准。突变可以使用下一代测序（NGS）或实时 PCR 为基础的检测方法进行评估。NGS 的结果时间为 1 到 2 周，而实时 PCR 为基础的检测方法的结果时间为 1 到 3 天。使用 NGS 可能会延迟一线治疗的开始，而使用基于 PCR 的检测方法受到可分析目标数量的限制。Idylla 系统是一种实时 PCR 盒式检测系统，能够使用 FFPE 肿瘤组织样本的一个部分分析热点突变。为了结合较短的延迟和大基因面板的分析，我们在此提出了一种实验室工作流程，该流程结合了 Idylla 系统和 NGS，并与低组织量的 FFPE 样本兼容。在这项研究中，我们评估并验证了通过直接将 DNA 吸入盒式系统而不是按照制造商建议的将 FFPE 部分吸入盒式系统的方式，使用 Idylla 系统对 RAS 和 BRAF 突变进行分析的方法。
从 29 例经 NGS 鉴定为 RAS 和 BRAF 突变的 mCRC 患者的 FFPE 样本中提取 DNA，并用 Idylla KRAS 和 Idylla NRAS-BRAF 突变检测试剂盒对其进行检测，以评估每种检测方法的敏感性、特异性、重复性和检测限。
对于 KRAS（12/12）、NRAS（12/12）和 BRAF（11/11）突变的测定，NGS 和 Idylla 结果之间存在 100%的一致性，敏感性和特异性均为 100%。该系统具有良好的可重复性，CV 低于 3%。KRAS 和 NRAS 突变的 LOD 达到 2.5ng 的 DNA，而 BRAF 突变的 LOD 达到 5ng 的 DNA。
使用直接将 DNA 吸入 Idylla 系统盒式系统的方法分析 RAS 和 BRAF 突变，显示出良好的敏感性、特异性、重复性和检测限，并且可以整合到具有少量组织的实验室工作流程中，而不会影响使用 NGS 对肿瘤进行进一步的全面特征分析。
