泛肿瘤基因突变检测高通量测序基因包的开发及临床验证  

Development and clinical validation of a high-throughput next-generation sequencing gene panel for pan-tumor mutations detection

作　　者：张晓密 梁志坤 方鹏 陈思远 周南 蒋析文 ZHANG Xiaomi;LIANG Zhikun;FANG Peng;CHEN Siyuan;ZHOU Nan;JIANG Xiwen(Guangdong Pharmaceutical University,Guangdong Guangzhou 510006,China;Daan Gene Co.,Ltd.,Guangdong Guangzhou 510665,China)

机构地区：[1]广东药科大学,广东广州510006 [2]广州达安基因股份有限公司研究院,广东广州510665

出　　处：《现代肿瘤医学》2024年第4期623-628,共6页Journal of Modern Oncology

基　　金：广东省广州市2023年度重点研发计划农业和社会发展科技专题项目(编号:2023B03J1381)。

摘　　要：目的:本研究旨在开发高通量测序基因包(panel)以实现个性化的泛肿瘤突变基因的检测,同时验证其临床适用性。方法:通过公开数据库选择并设计223个肿瘤相关基因的引物组成panel,并以菁良公司的不同百分比结构变异福尔马林固定石蜡包埋(formalin-fixed paraffin-embedded,FFPE)标准品、野生型标准品、泛肿瘤800标准品、Horizon Discovery公司的游离DNA标准品为样本完成本panel准确性、重复性和检测限的实验室验证,并利用10例组织样本和2例游离DNA样本进行临床验证。结果:在准确性上,本panel阳性和阴性检出符合率达到100%。在重复性上,利用5%结构变异FFPE标准品具有良好的重复性,而游离DNA标准品重复性一般。在最低检测限上,2.5%结构变异FFPE标准品可稳定检出,最低可检出1%结构变异标准品;游离DNA标准品的最低检测限为0.25%。利用本panel检测临床样本表现出与对照方法良好的一致性。结论:成功开发靶向针对223个肿瘤相关基因的高通量测序panel并完成了分析性能和临床适用性验证,该方法具有协助临床诊断与治疗的潜力。Objective:To develop a high-throughput sequencing gene panel for the detection of personalized pan-cancer mutation genes and validate its clinical applicability.Methods:A total of 223 tumor-associated genes were selected based on public databases and primer sets were designed for the gene panel.Laboratory validation of the analytical performance,including accuracy,repeatability,and detection limit,was conducted using GeneWell formalin-fixed paraffin-embedded(FFPE)reference standards,wild-type standards,pan-cancer 800 reference standards,and Horizon Discovery cfDNA reference standards.Clinical validation was performed using 10 tissue samples and 2 cfDNA samples.Results:The gene panel exhibited 100% concordance for both positive and negative detection in terms of accuracy.Repeatability was shown to be excellent using the FFPE reference standard containing 5% structural variant,while the cfDNA reference standard showed moderate repeatability.The gene panel achieved stable detection at a minimum level of 2.5%,with a minimum detectable limit of 1%.The lowest detectable limit for the cfDNA reference standard was 0.25%.The gene panel demonstrated good consistency with the control method when applied to clinical samples.Conclusion:In this study,we successfully developed a high-throughput gene panel for 223 tumor-associated genes and validated its analytical performance and clinical applicability.This method has been proven to have the potential to assist clinical diagnosis and treatment.

关 键 词：高通量测序 基因包 泛肿瘤 基因突变 

分 类 号：R73-3[医药卫生—肿瘤]