结直肠癌患者血浆cfDNA检测方法的建立及临床应用评估

Establishment and clinical application evaluation of plasma cfDNA determination in colorectal cancer patients

目的建立基于下一代测序(NGS)平台的检测结直肠癌患者血浆循环游离DNA(cfDNA)的方法,并进行性能确认和临床应用评价。方法在NGS平台上构建测定结直肠癌患者血浆cfDNA的检测系统(简称结直肠癌cfDNA panel)。对结直肠癌cfDNA panel的准确性、精确性、可用于抽提的最少样本量和投入量、分析灵敏度、分析特异性等性能指标进行评估。比较结直肠癌cfDNA panel与Miseq Dx二代测序平台及突变扩增阻滞系统(ARMS)(肿瘤组织样本)的一致性。结果结直肠癌cfDNA panel的准确性为99.97%,对预期突变的检出率为100%。可用于血浆抽提的样本起始量为1 mL,cfDNA投入量为10 ng,分析灵敏度为0.2%。10000 mg/L血红蛋白、500 mg/L胆红素或2%脂肪乳对结直肠癌cfDNA panel抽提无影响。结直肠癌cfDNA panel与Miseq Dx二代测序平台和ARMS(肿瘤组织样本)的一致性分别为94.12%和90.91%,结直肠癌cfDNA panel与Miseq Dx二代测序平台检测KRAS、NRAS、BRAF、PIK3CA的阳性符合率和阴性符合率分别为100%和99.60%。结论结直肠癌cfDNA panel的检测性能符合临床应用要求,且血浆cfDNA结果与肿瘤组织结果高度一致,可作为组织活检的有益补充。

Objective To establish and evaluate the performance and clinical application of plasma cfDNA determination in colorectal cancer patients based on next generation sequencing(NGS)platform.Methods Plasma circulating free DNA(cfDNA)determination system(cfDNA panel)for colorectal cancer patients had been established.The accuracy,precision,minimum sample amount and input of extraction,sensitivity and specificity of cfDNA panel were evaluated.The results were compared with both the results of Miseq Dx NGS platform and amplification refractory mutation system(ARMS).Results The accuracy of cfDNA panel for colorectal cancer was 99.97%,and the determination rate of expected mutation was 100%.The minimum amount of plasma extraction was 1 mL,and the minimum amount of cfDNA input was 10 ng.The sensitivity was 0.2%.The 10000 mg/L hemoglobin,500 mg/L bilirubin or 2%fat emulsion had no effet on the extraction of colorectal cancer cfDNA panel.Compared with the results of Miseq Dx NGS platform and ARMS,the consistency rates were 94.12%and 90.91%,respectively,and the positive and negative predictive rates of KRAS,NRAS,BRAF and PIK3CA were 100%and 99.60%,respectively.Conclusions The performance of cfDNA panel in colorectal cancer meets the needs of clinical application.The results of plasma cfDNA determination are highly consistent with the determination results of matched tumor tissue,which is a useful supplement for tissue biopsy.