Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
Department of Laboratory Medicine, Netherlands Cancer Institute, Amsterdam, the Netherlands.
Clin Chem. 2024 May 2;70(5):759-767. doi: 10.1093/clinchem/hvae014.
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).
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.
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.
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.
在循环肿瘤 DNA（ctDNA）中鉴定肿瘤衍生变体具有作为肿瘤组织基础常规诊断检测的敏感和可靠替代物的潜力。然而，前（分析前）程序的变化会影响 ctDNA 回收的效率。在此，进行了一项外部质量评估（EQA），以确定在荷兰 COIN 联盟（ctDNA 在荷兰实施的道路上）内的实验室当前诊断设置中使用的 ctDNA 突变检测工作流程的性能。
将 3 份高容量诊断白细胞分离（DLA）血浆样本和 3 份具有预定突变的人工参考血浆样本的等分试样分发给 16 个荷兰实验室。要求参与实验室使用其常规循环无细胞 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、总体检测率和总体基因分型性能对实验室进行评估。
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，液滴数字 PCR [ddPCR]、小面板 PCR 测定和下一代测序 [NGS]）。6 个实验室（38%）的表现得分为>0.90；其他所有实验室的得分在 0.26 至 0.80 之间。尽管 13 个实验室（81%）达到了 100%的总体检测率，但具有治疗意义的 EGFR p.(S752_I759del)（69%）、EGFR p.(N771_H773dup)（50%）和 KRAS p.(G12C)（48%）突变经常无法准确地进行基因分型。
使用相同的血浆样本时，不同的（分析前）协议可能导致不同的临床结果。（分析前）工作流程的标准化可以促进可重复的液体活检测试在临床常规中的实施。
