Abstract 3491: Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR.
Authors: Kristof Jessica , Bruening Eric , Wong Stephane , Sankar Sabita 
Published: August 2014

Background: Non-small cell lung cancer (NSCLC) patients who harbor activating mutations in epidermal growth factor receptor (EGFR) such as L858R and the Exon 19 deletions (E746-A750) are treated with either erlotinib or gefitinib. However, a large percentage of patients acquire the gatekeeper resistance mutation, T790M. Limited access to post-treatment tumor tissue restricts monitoring options, and even when biopsy specimens are available, sampling bias may confound diagnosis of the resistance mechanism. Circulating, cell-free DNA (CNA) provides a solution to circumvent tissue access limitations. Low overall recovery of CNA requires assays be designed with increased sensitivity and specificity not currently available. We have developed and optimized a series of droplet digital PCR assays that can quantify both EGFR activating and resistance mutations as well as copy number.

Methods: The ddPCR assays were developed on the BioRAD QX100 system. Primers and probes were designed to optimize quantification of EGFR L858R and T790M mutations, Exon 19 deletions and EGFR copy number. Genomic DNA from H1975 (L5858R and T790M mutations) and H1650 (Exon 19 deletion) cell lines were diluted into wild type gDNA to determine the assays’ limits of detection (LOD). Patient samples were analyzed as well. Finally, ddPCR data were compared with qPCR data using the Qiagen EGFR Rotor-Gene (RGQ) kit.

Results: The ddPCR assays were designed to quantify EGFR activating and resistance mutations in relative to the wild type template. The T790M and L858R assays displayed linear dynamic ranges from ∼14000 copies to LODs of 9 and 6 copies, respectively. In addition, ddPCR assays were successfully multiplexed to an LOD of 0.2%, or 15 copies based upon a 95% confidence interval in 20ng gDNA. In healthy donor spiked plasma samples, the LOD was determined to be 1.2%, or 8 copies in 2.2ng purified CNA. By comparison, in plasma, the RGQ assays displayed an average LOD of 80 copies with a dynamic range of ∼8000 copies to 76 copies. Data on Exon 19 deletion and EGFR copy number ddPCR assays will be presented. Spiked normal plasma samples analyzed for the ratio of T790M to activating mutations such as L858R and Exon 19 deletion will be presented.

Conclusion: Droplet digital PCR provides sensitive quantification of low abundance EGFR resistance and activating mutations in CNA isolated from plasma. These assays provide a useful method for selecting treatment, monitoring disease progression, and providing early detection of treatment failure as well as gene amplification associated with EGFR acquired resistance.

Citation Format: Jessica Kristof, Eric Bruening, Stephane Wong, Sabita Sankar. Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2013-3491