Background: Liquid biopsy enables the non-invasive analysis of genetic tumor variants in circulating free DNA (cfDNA) in plasma. Accurate analytical validation of liquid biopsy NGS assays is required to detect variants with low variant allele frequencies (VAFs). Methods: Six types of commercial cfDNA reference materials and 42 patient samples were analyzed using a duplex-sequencing-based liquid biopsy NGS assay. Results: We comprehensively evaluated the similarity of commercial cfDNA reference materials to native cfDNA. We observed significant differences between the reference materials in terms of wet-lab and sequencing quality as well as background noise. No reference material resembled native cfDNA in all performance metrics investigated. Based on our results, we established guidelines for the selection of appropriate reference materials for the different steps in performance evaluation. The use of inappropriate materials and cutoffs could eventually lead to a lower sensitivity for variant detection. Conclusion: Careful consideration of commercial reference materials is required for performance evaluation of liquid biopsy NGS assays. While the similarity to native cfDNA aids in the development of experimental protocols, reference materials with well-defined variants are preferable for determining sensitivity and precision, which are essential for accurate clinical interpretation.
背景：液体活检技术能够通过血浆中循环游离DNA（cfDNA）非侵入性地分析肿瘤基因变异。为检测低变异等位基因频率（VAF）的变异，需要对液体活检二代测序（NGS）检测方法进行精确的分析验证。方法：采用基于双链测序的液体活检NGS检测方法，对六种商业cfDNA参考物质及42例患者样本进行分析。结果：我们全面评估了商业cfDNA参考物质与天然cfDNA的相似性。研究发现，不同参考物质在湿实验质量、测序质量及背景噪音方面存在显著差异。在所有考察的性能指标中，未发现任何一种参考物质能在所有方面与天然cfDNA完全相似。基于研究结果，我们制定了针对性能评估不同阶段选择合适参考物质的指导原则。使用不合适的参考物质及判定阈值可能导致变异检测灵敏度降低。结论：液体活检NGS检测的性能评估需审慎选择商业参考物质。虽然与天然cfDNA的相似性有助于实验方案的开发，但具有明确变异特征的参考物质更适用于确定检测灵敏度与精密度，这对临床准确解读检测结果至关重要。
Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
肿瘤(癌症)患者之家