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Beili Wang, Shengchao Wu, Fei Huang, Minna Shen, Huiqin Jiang, Yiyi Yu, Qian Yu, Yihui Yang, Ying Zhao, Yiwen Zhou, Baishen Pan, Tianshu Liu, Wei Guo
Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, P.R. China.
Department of Medical Oncology, Center of Evidence Based Medicine, Zhongshan Hospital, Fudan University, Shanghai, P.R. China.
Clin Chem Lab Med. 2019 Sep 25;57(10):1501-1510. doi: 10.1515/cclm-2019-0142.
Background Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy. Methods Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively. Conclusions Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.
背景 评估肿瘤 RAS/BRAF 状态对于转移性结直肠癌（mCRC）患者的治疗选择和预后评估很重要。纠正文库制备和测序中的伪影对于准确分析循环肿瘤 DNA（ctDNA）突变至关重要。在这里，我们评估了一种新型基于扩增子的下一代测序（NGS）检测方法 Firefly™的分析和临床性能，该方法采用串联体纠错策略。 方法 使用无细胞 DNA（cfDNA）参考标准和 184 例 mCRC 患者的 cfDNA 样本评估 Firefly 检测针对 KRAS/NRAS/BRAF/PIK3CA 的检测性能。将血浆结果与来自匹配组织的基于 ARMS 的 PCR 确定的突变状态进行比较。将突变丰度低于检测限（LOD）的样本再次通过液滴数字聚合酶链反应（ddPCR）或 NGS 进行重新检测。 结果 Firefly 检测法在 20ng cfDNA 时，等位基因频率（AF）为 0.2%时的检测率达到 98.89%，显示出优异的灵敏度和特异性。通常，通过 NGS 检测血浆 cfDNA 和 ARMS 检测 FFPE 组织，分别有 40.76%和 48.37%的患者被报告为阳性。两种平台之间的一致性率为 80.11%。在治疗前队列中，根据 Firefly™和 ARMS 基因分型的 17 个常见外显子，血浆和组织之间的一致性率为 93.33%，KRAS/NRAS/BRAF/PIK3CA 的阳性符合率（PPA）和阴性符合率（NPA）均为 100%和 99.60%。 结论 Firefly 检测到的总血浆 cfDNA 为 CRC 患者的突变谱分析提供了可行的补充，因为与匹配的肿瘤样本具有很高的一致性。总之，这些数据表明 Firefly 可常规应用于 mCRC 患者的临床应用。
