Background Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). Methods Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18–21, and KRAS exon 2–3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. Results A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. Conclusions Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.

中文翻译：

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使用 COIN 联盟内临床病理学常规使用的基于循环肿瘤 DNA 的方法对分子肿瘤分析进行外部质量评估

背景 循环肿瘤 DNA (ctDNA) 中肿瘤衍生变异的鉴定有可能作为基于肿瘤组织的常规诊断测试的敏感且可靠的替代物。然而，预（分析）程序的变化会影响 ctDNA 回收的效率。在这里，进行了外部质量评估 (EQA)，以确定 ctDNA 突变检测工作流程的性能，该工作流程用于荷兰 COIN 联盟内各个实验室的当前诊断设置（ctDNA 正在荷兰实施）。方法 将 3 份大容量诊断白细胞分离术 (DLA) 血浆样本和 3 份具有预定突变的人工参考血浆样本等分试样分发给 16 个荷兰实验室。要求参与实验室使用其常规循环游离 DNA (ccfDNA) 分析工作流程对 BRAF 外显子 15、EGFR 外显子 18-21 和 KRAS 外显子 2-3 进行 ctDNA 分析。根据对研究方案的遵守情况、总体检出率和总体基因分型表现对实验室进行评估。结果 使用了广泛的分析前条件（例如血浆体积、洗脱体积和提取方法）和分析方法（例如微滴数字 PCR [ddPCR]、小板 PCR 检测和新一代测序 [NGS]） 。六个实验室（38%）的绩效得分>0.90；所有其他实验室得分在 0.26 到 0.80 之间。尽管 13 个实验室 (81%) 的总体检出率达到 100%，但治疗相关的 EGFR p.(S752_I759del) (69%)、EGFR p.(N771_H773dup) (50%) 和 KRAS p.(G12C) (48%) ）突变常常无法准确地进行基因分型。结论 使用相同血浆样本时，不同的（预）分析方案可能会导致不同的临床结果。（预）分析工作流程的标准化可以促进在临床常规中实施可重复的液体活检测试。