摘要：

Lung cancer is the most common cause of global cancer-related mortality, leading to over a million deaths each year, with adenocarcinoma the most common histological type. Recently, molecularly targeted therapies have dramatically improved the treatment of patients whose tumors are driven by somatically activated oncogenes such as mutated EGFR, BRAF or ERBB2, or translocated ALK, RET, or ROS1. In addition, circulating cell-free DNA in the blood is undergoing assessment as an accessible biomarker for dynamic monitoring of treatment efficacy and clonal evolution of the cancer patient's entire tumor burden. Thus a new paradigm for monitoring and resistance of solid tumors is currently under development: sequencing the initial tumor biopsy for driver mutations followed by ultrasensitive detection of circulating tumor DNA (ctDNA) using methods such as digital PCR (dPCR). In recent years, dPCR has become the gold standard for precise quantification of nucleic acids, showing detection sensitivities unparalleled by other methodologies. Picodroplet-formatted dPCR compartmentalizes the sample (and one or more assay) into a large enough number of discrete reaction volumes (e.g. 10 million) such that there is at most one target molecule per reaction. Standard qPCR reagents (target-specific primers and fluorescent hydrolysis probes) and master mixes are used for endpoint PCR amplification, resulting in plateaued fluorescent signals only in target-containing compartments. This enables digital counting of the absolute number of target molecules present in the sample volume tested. Here we report the use of the RainDrop dPCR system together with multiplexed assays measuring the most common cancer-relevant mutations in EGFR. Multiplexed primer and probe sets were designed for wild-type EGFR, T790M and L858R point mutations, and exon 19 deletions in EGFR. Using spike-in plasmid controls and genomic DNA from lung cancer cell lines (H1975,H1650), we verified simultaneous detection and discrimination of all mutations across a broad dynamic range. Finally, we demonstrated application of the multiplexed EGFR assays on matched tumor and ctDNA samples from lung cancer patients. Sensitive quantification of EGFR mutations in lung cancer patients is critical for stratifying patients into different treatment regimens, and can potentially benefit EGFR mutation-positive patients by allowing dynamic non-invasive tracking of treatment efficacy and clonal evolution. Development of a multiplexed dPCR EGFR mutation assay should facilitate personalizing lung cancer therapy and treatment management, towards the goal of monitoring disease progression for better outcomes. The RainDrop system enables sensitive simultaneous dPCR detection of multiple mutations from the same sample, which is particularly important when available sample is limited, e.g. with tumor biopsies, FFPE samples, circulating tumor cells, and ctDNA in biological fluids. Citation Format: Michael L. Samuels, Christina Read, Jennifer Jackson, Damien Slater, David Chappell, Mark Consugar. Multiplex digital PCR analysis of EGFR mutations for lung cancer characterization and monitoring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3647.

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