Abstract B072: Liquid biopsy analysis of NSCLC patient plasma using ddPCR and NGS
Authors: Liu WeiHua , Fang Peng , Galderisi Chad , Spittle Cindy , Li Jin 
Published: August 2018

Abstract
Introduction: Analysis of cfDNA has potential applications in therapy selection and disease monitoring in the clinical management of NSCLC. Here we use EGFR ddPCR and the Oncomine Lung cfDNA Assay to evaluate the mutation profile and allele burden in a set of plasma samples collected from NSCLC patients. Methods: Plasma samples were collected at Dana-Farber Cancer Institute (DFCI). cfDNA was extracted using the QIAamp DSP circulating NA kit. EGFR ddPCR assays were designed by DFCI. One third of cfDNA extracted from 1 ml plasma was used for each EGFR ddPCR assay (L858R, T790M and ex19del). The L858R and T790M ddPCR assays detect mutations using mutant specific probes. The ex19del assay allows for the detection of a broad range of exon 19 deletions via the loss of wild-type exon 19 signal. Twenty-two plasma samples from patients with EGFR WT tumors were analyzed to assess the background noise (Limit of Blank). The other 32 plasma samples were analyzed in a blinded fashion. ddPCR analysis was performed on the QX200 system and data was analyzed using QuantaSoft software. A subset of 12 plasma samples had sufficient cfDNA for additional analysis using the Oncomine Lung cfDNA Assay on the Ion S5. This NGS panel is designed to detect 150 hotspot mutations in 11 lung cancer-related genes. Torrent Suite Software 5.2 was used for NGS data analysis. The HorizonDx cfDNA Multiplex Reference Standard (AF 5%, 1%, 0.1%, and WT) was also included in the ddPCR and NGS studies. Results: Based on the Limit of Blank data, the positive mutation cut-off for each EGFR ddPCR assay was set at 3 mutant copies with minimum 3000 wild type copies. Studies using the HorizonDx cfDNA standard demonstrated that the analytical sensitivity of the ddPCR assays increases with cfDNA input: the lowest detected allele frequency (AF) is 1%, 0.5%, and 0.25% for 3, 6, and 12ng input, respectively. In plasma samples, EGFR mutations were detected as low as 0.08% AF using ddPCR (4.5 L858R mutant copies/5827 wild type copies). L858R and exon 19 deletions were detected in 13/54 and 21/54 plasma samples, respectively. T790M mutations were detected in 19/54 plasma samples, and coexisted with L858R or ex19del in all cases. The Oncomine™ Lung cfDNA Assay showed 100% concordance with EGFR ddPCR results for the 12 plasma samples analyzed by both methods. Mutations were detected as low as 0.18% AF with 8.5ng plasma cfDNA input in the NGS assay. Using the HorizonDx cfDNA multiplex standard mutations were detected as low as 0.1% AF using 20ng input. In addition, PIK3CA mutations were found to coexist with EGFR ex19 deletions in 3/12 plasma samples using NGS. The AF of PIK3CA mutations and EGFR mutations was comparable in the same samples (TRL23: 24.3% for EGFR del19 and 10.4% T790M vs 20.2% for PIK3CA H1047R; TRL48: 7.8% for EGFR del19 vs 3.4% for PIK3CA E545K; TRL51: 0.85% for EGFR del19 vs 1.02% for PIK3CA E545K). Conclusions: Both ddPCR and NGS were able to detect mutations at 0.1% AF with sufficient cfDNA input. The Oncomine panel confirmed EGFR ddPCR results and detected coexisting PIK3CA mutations. EGFR and PIK3CA mutations were present at a similar AF, suggesting that EGFR and PIK3CA mutations may develop at the same stage of tumorigenesis in these patients. Our study results demonstrate that comprehensive profiling of cfDNA using NGS could be critical for the selection of appropriate therapies for NSCLC patients while ddPCR is an ideal platform for monitoring disease burden during the course of therapy.
Citation Format: WeiHua Liu, Peng Fang, Chad Galderisi, Cindy Spittle, Jin Li. Liquid biopsy analysis of NSCLC patient plasma using ddPCR and NGS [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B072.