Abstract 3490: Highly sensitive detection of EGFR T790M on Ion Torrent PGM.
Authors: Darwanto Agus , Fang Peng , Yan Zhenyu , Liu Weihua , Pelak Kim , Kristof Jessica , Sankar Sabita , Spittle Cynthia , Galderisi Chad , Li Jin 
Published: August 2014

Abstract
Introduction: EGFR T790M mutation leads to treatment resistance in ∼50% of NSCLC patient undergoing TKI treatment. Early detection of the emergence of this resistance mutation allows for tailoring of the treatment regimen. Detection of EGFR T790M requires higher sensitivity techniques than those needed to detect EGFR activating mutations, and quantitative measurement of T790M may also provide value in monitoring disease progression. Here we demonstrate that our proprietary amplicon next-generation sequencing (NGS) on Ion Torrent PGM provides an attractive solution for T790M detection with the advantage of high sensitivity, specificity, and quantification capability.
Methods: We developed a proprietary library prep method for amplicon sequencing on the Ion Torrent PGM. Ion Torrent barcode sequences were refined in our validation to minimize barcode cross contamination. This optimization of barcodes allows for accurate mutation quantification, especially critical for variants with a frequency below 1%. This also added the benefit of higher throughput and lower cost. We employ this non-enrichment method to quantitatively measure T790M.
DNA from the NCI-H1975 cell line was serially diluted into wild-type NA19240 DNA to determine the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility of the method. We also used reference FFPE DNA from HorizonDx to validate the accuracy and robustness of the assay. We further validated the test by comparing the performance of our NGS method with ddPCR using the same sample set (serially diluted H1975 into wild-type DNA controls).
Results: Our data demonstrated that the LOD for T790M is 0.2%. And the method could reproducibly identify variants at these frequencies. With an input of 10ng of the HorizonDx FFPE DNA, we measured T790M frequency at 6.7%, which is concordant with the 6.5% mutation frequency reported by HorizonDx. We cross-validated our NGS assay with ddPCR, and the mutation frequencies detected by both platforms are nearly identical, and have a regression coefficient of 0.9995, with comparable LOD of 0.2%. Furthermore, our NGS assay allows us to detect other variants located in this amplicon, e.g. SNP Q787Q, with LOD at 1%. The phasing status of compound mutations can also be determined by this assay.
To further improve the sensitivity, an enrichment method was developed, and the enriched T790M was sequenced by PGM. This method can semi-quantitatively measure T790M frequency with LOD of 0.03%.
Conclusion: Our EGFR T790M NGS assay provides a unique option to reliably quantify T790M down to 0.2% with low DNA input. This highly sensitive and specific detection capability may enable earlier detection of emerging therapeutic resistance, particularly if FFPE results reported here extend to a more accessible specimen type, i.e. plasma circulating DNA, amenable to periodic patient monitoring.
Citation Format: Agus Darwanto, Peng Fang, Zhenyu Yan, Weihua Liu, Kim Pelak, Jessica Kristof, Sabita Sankar, Cynthia Spittle, Chad Galderisi, Jin Li. Highly sensitive detection of EGFR T790M on Ion Torrent PGM. [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 3490. doi:10.1158/1538-7445.AM2013-3490