PO.CL01.04 · 临床研究
ddPLEX EGFR/KRAS/BRAF：一种用于超灵敏NSCLC生物标志物检测的高度多重微滴式数字PCR(ddPCR)panel
ddPLEX EGFR/KRAS/BRAF: A highly multiplexed droplet digital PCR (ddPCR) panel for ultra-sensitive NSCLC biomarker detection
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
基于在符合条件的转移性NSCLC患者中使用靶向治疗所观察到的结局改善，国家和国际指南推荐针对可干预的致癌驱动突变进行生物标志物检测。在此，我们展示ddPLEX EGFR/KRAS/BRAF突变检测分析试剂盒的性能，该试剂盒为仅供研究使用的试剂盒，专为QX600™微滴式数字™PCR(ddPCR)系统开发。该试剂盒旨在于单个ddPCR孔中检测、区分并定量EGFR、KRAS和BRAF基因中的37个可干预变异。它还配有一个总量定量孔，以突变不可知的方式定量EGFR、KRAS和BRAF基因，并可计算变异等位基因频率(VAF)。它通过简化的工作流程提供当日结果，包含阳性对照和内部对照，并兼容血浆和福尔马林固定石蜡包埋(FFPE)样本。
ddPLEX EGFR/KRAS/BRAF突变检测分析试剂盒的分析性能已在内部进行评估，其结果呈现于此。结果表明，对于所有靶向变异，在132 ng人类基因组DNA背景下分析灵敏度为0.025%-0.1%。当以每个变异1000拷贝输入逐一测试(共132 ng人类基因组DNA)时，该分析的分析特异性为100%。突变分析未与位于KRAS G12C和BRAF V600E同一扩增子内的任何常见报道突变发生交叉反应。突变分析在每反应800拷贝至4拷贝范围内呈线性，R²在0.94-0.99范围内。总量定量分析在1.3 ng至53 ng总DNA输入范围内亦呈线性，三个靶标的R²均>0.99。对于既往提取并存档、且已用其他基于PCR方法检测过的血浆游离DNA和FFPE DNA，ddPLEX试剂盒的一致性分别为100%和96.5%。
总之，ddPLEX EGFR/KRAS/BRAF突变检测分析试剂盒在检测临床相关的NSCLC变异方面灵敏、准确且在时间和成本上高效。多重化最大限度地提高信息量，并减少来自FFPE和血浆等核酸量低的标本的取样偏差。
查看英文原文 English abstract
Biomarker testing for actionable oncogenic driver mutations is recommended in national and international guidelines based on the improved outcomes observed with use of targeted therapies in eligible patients with metastatic NSCLC. Here, we demonstrate the performance of the ddPLEX EGFR/KRAS/BRAF Mutation Detection Assay Kit, a research use only kit developed for the QX600 TM Droplet Digital TM PCR (ddPCR) System. This kit is designed for the detection, discrimination and quantification of 37 actionable variants in the EGFR , KRAS , and BRAF genes in a single ddPCR well. It also has a companion total quantification well that quantifies EGFR , KRAS and BRAF genes in a mutation agnostic manner and allows for variant allele frequency (VAF) calculation. It provides same day results with a streamlined workflow, includes positive and internal controls and is compatible with plasma and formalin-fixed paraffin-embedded (FFPE) samples.
The analytical performance of the ddPLEX EGFR/KRAS/BRAF Mutation Detection Assay Kit was evaluated internally, and the findings are presented here. The results demonstrated an analytical sensitivity of 0.025%-0.1% in a background of 132 ng of human genomic DNA for all targeted variants. The assay had an analytical specificity of 100% when tested with 1000 copies input of each variant, one at a time, in a total of 132 ng of human genomic DNA. The mutant assay did not cross-react with any of the commonly reported mutations located in the same amplicon as KRAS G12C and BRAF V600E. The mutant assay was linear from 800 copies to 4 copies per reaction with a R 2 in the range of 0.94-0.99. The total quantification assay was also linear in the range of 1.3 ng to 53 ng of total DNA input with a R 2 >0.99 for all three targets. With previously extracted and archived cell-free DNA from plasma and FFPE DNA that were tested with alternative PCR based methods, the ddPLEX kit had a concordance of 100% and 96.5%, respectively.
In conclusion, the ddPLEX EGFR/KRAS/BRAF Mutation Detection Assay Kit is sensitive, accurate, and efficient in time and cost for detecting clinically relevant NSCLC variants. Multiplexing maximizes information and reduces sampling bias from specimens with low nucleic acid quantities such as FFPE and plasma.
利益披露 Disclosure
S. Jain, None..
C. Festog, None..
M. Ho, None..
C. Li, None..
N. Mehmet, None..
A. Melendez-Molina, None..
T. Newsum, None..
P. Pal, None..
M. Rostamza, None..
Y. Ono, None..
L. Jackson, None..
B. D’Alessio, None..
G. Johnson, None..
G. A. Pestano, None.