Mehrotra Meenakshi, Singh Rajesh R, Loghavi Sanam, Duose Dzifa Yawa, Barkoh Bedia A, Behrens Carmen, Patel Keyur P, Routbort Mark J, Kopetz Scott, Broaddus Russell R, Medeiros L Jeffrey, Wistuba Ignacio I, Luthra Rajyalakshmi
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Oncotarget. 2017 Oct 24;9(12):10259-10271. doi: 10.18632/oncotarget.21982. eCollection 2018 Feb 13.
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; < 0.03) and cfDNA negative for mutations (74.2 vs 53 months; < 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.
需要一个合适的临床级平台来在实体瘤患者的游离DNA（cfDNA）中高灵敏度地检测体细胞突变。在本研究中，我们同时评估了用于cfDNA基因分型的超深度NGS与MassARRAY和等位基因特异性液滴数字PCR（ddPCR），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Ion Proton上的Ampliseq癌症热点面板V2，通过深度测序评估了46例患有各种晚期转移性实体瘤且已知突变的患者的血浆样本。对这些有DNA可用的样本子集进行了ddPCR和UltraSEEK MassARRAY检测，以检测5个基因（IDH1、PIK3CA、KRAS、BRAF和NRAS）中的突变。在cfDNA中检测到了104个预期组织突变中的61个以及组织中不存在的另外6个突变。ddPCR和MassARRAY与NGS在突变检测方面的一致性分别为83%和77%，灵敏度分别为100%和79%。cfDNA产量较低的患者（74对50个月；<0.03）和cfDNA突变阴性的患者（74.2对53个月；<0.04）的中位OS明显长于cfDNA产量较高且突变阳性的患者。使用系列稀释的阳性cfDNA样本，证明ddPCR和MassARRAY平台的检测限为0.1%。MassARRAY和ddPCR系统能够对一组靶向突变进行快速且经济高效的基因分型，可用于单基因检测以指导化疗反应或对NGS结果进行正交验证。
