摘要目的 评估结直肠癌患者肠镜活检标本中的DNA是否足以进行基于扩增法建库的二代测序(NGS)检测以得到合格、准确的基因分析结果并对甲醛固定石蜡包埋(FFPE)小组织行NGS检测提出质控建议.方法 依据标本纳入标准收集2012年1月至2016年6月四川大学华西医院和北京大学第三医院组织病理学确诊的结直肠癌109例患者的肠镜活检FFPE标本109份,提取DNA,采用OncoAimTMGene Panel在Miseq平台进行基于扩增法建库的靶向NGS检测,分析DNA及文库产量、测序数据质量、检测成功率.利用常规检测方法验证NGS的检测结果.最后,比较其中12个内镜活检标本与其相应手术根治石蜡标本的 NGS 检出突变结果以评估检测结果的一致性.结果97.2%(106/109)的肠镜活检FFPE标本可得到足量用于NGS的DNA并产生合格的测序文库,测序深度中位数为848×,均一度中位数为95.7%,测序数据合格率为95.4%(104/109). NGS和临床常规检测的一致性为100.0%. 12对内镜活检与手术根治配对标本结果显示,临床可行性突变( KRAS、BRAF)的检测结果全部一致,而一致突变的变异频率均有明显差异.结论 合理质控的条件下,肠镜活检FFPE标本可用于靶向NGS检测,得到合格、准确的突变检测结果以指导靶向治疗.

abstractsObjective To investigate whether small endoscopic biopsies of colorectal cancer were sufficient for quality and accurate mutational analysis by amplicon-based next-generation sequencing (NGS). Methods By using an amplicon-based targeted NGS panel for mutational detection on Illumina Miseq platform, a total of 109 formalin-fixed and paraffin-embedded ( FFPE) endoscopic biopsies of colorectal cancer were retrospectively selected, based on specific histopathologic criteria, from January 2012 to June 2016 at West China Hospital of Sichuan University and Peking University Third Hospital. Twelve of these biopsies had corresponding FFPE surgical resection specimens. Quality control parameters of NGS testing were analyzed and NGS results were confirmed by other methods. Mutation calls of the 12 paired endoscopic biopsies and surgical resections were compared. Results Of the endoscopic biopsy specimens, 97.2%(106/109) had sufficient DNA and qualified sequencing library. NGS generated excellent sequencing data, with a median of 848× for median read depth and 95.7% for uniformity. The success rate of NGS was 95.4%(104/109). Conventional methods confirmed the results of NGS for KRAS and BRAF, and the concordance rate was 100.0%. The clinically actionable mutations detected in the 12 paired endoscopic biopsies and surgical resections were concordant. Conclusion FFPE endoscopic biopsies of colorectal cancer is suitable for targeted NGS, providing quality sequencing data and accurate mutational information to guide targeted therapy.