Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors.
Detection of somatic mutations in cell-free DNA in plasma and correlation with overall survival in patients with solid tumors.
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DOI：
10.18632/oncotarget.21982
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发表时间：
2018-02-13
期刊：
影响因子：
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通讯作者：
Luthra R
中科院分区：
文献类型：
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作者：
Mehrotra M;Singh RR;Loghavi S;Duose DY;Barkoh BA;Behrens C;Patel KP;Routbort MJ;Kopetz S;Broaddus RR;Medeiros LJ;Wistuba II;Luthra R
为了检测实体瘤患者的无细胞DNA(CfDNA)中高灵敏度的体细胞突变，需要一个合适的临床级平台。在这项研究中，我们评估了并行的超深NGS、MassARRAY和等位基因特异性滴状数字聚合酶链式反应(DdPCR)在cfDNA分型中的作用，并将cfDNA产量和突变状态与患者的总生存期(OS)相关联。我们使用离子质子上的AmpliSeq癌症热点小组V2通过深度测序评估了46名患有各种晚期转移性实体肿瘤和已知突变的患者的血浆样本。利用ddPCR和UltraSEEK Massarray对这些样本中具有DNA可用性的子集进行检测，以检测5个基因(IDH1、PIK3CA、KRAS、BRAF和NRAS)的突变。在cfDNA中检测到了104个预期组织突变中的61个和组织中不存在的6个额外突变。与NGS法检测突变的符合率分别为83%和77%，敏感度分别为100%和79%。CfDNA产率较低(74vs50个月；P<；0.03)和cfDNA阴性患者(74.2vs53个月；p<；0.04)的中位OS显著长于cfDNA产量较高且突变阳性的患者。使用连续稀释的阳性cfDNA样品，ddPCR和MassARRAY平台的检出限为0.1%。MassARRAY和ddPCR系统能够对一组目标突变进行快速、经济有效的基因分型，并可用于单基因测试，以指导对化疗的反应或对NGS结果进行正交验证。
A suitable clinical-grade platform is required for detection of somatic mutations with high sensitivity in cell-free DNA (cfDNA) of patients with solid tumors. In this study, we evaluated in parallel ultra-deep NGS with MassARRAY and allele-specific droplet digital PCR (ddPCR) for cfDNA genotyping and correlated cfDNA yield and mutation status with overall survival (OS) of patients. We assessed plasma samples from 46 patients with various advanced metastatic solid tumors and known mutations by deep sequencing using an Ampliseq cancer hotspot panel V2 on Ion Proton. A subset of these samples with DNA availability was tested by ddPCR and UltraSEEK MassARRAY for mutation detection in 5 genes (IDH1, PIK3CA, KRAS, BRAF, and NRAS). Sixty one of 104 expected tissue mutations and 6 additional mutations not present in the tissue were detected in cfDNA. ddPCR and MassARRAY showed 83% and 77% concordance with NGS for mutation detection with 100% and 79% sensitivity, respectively. The median OS of patients with lower cfDNA yield (74 vs 50 months; P < 0.03) and cfDNA negative for mutations (74.2 vs 53 months; p < 0.04) was significantly longer than in patients with higher cfDNA yield and positive for mutations. A limit-of-detection of 0.1% was demonstrated for ddPCR and MassARRAY platforms using a serially diluted positive cfDNA sample. The MassARRAY and ddPCR systems enable fast and cost-effective genotyping for a targeted set of mutations and can be used for single gene testing to guide response to chemotherapy or for orthogonal validation of NGS results.