Abstract 1715: EGFR mutational detection in ctDNA, Vortex-enriched CTCs and comparison to tumor tissue in non-small-cell-lung-cancer (NSCLC) patients
Authors: Liu Haiyan E. , Vuppalapaty Meghah , Lemaire Clementine A. , Wilkerson Charles , Crouse Steve C. , Goldman Jonathan W. , Sollier-Christen Elodie 
Published: July 2017

Abstract
Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutation, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks and expense and are not always successful. In order to identify the development of secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKI’s and an indication for therapy with a 3rd generation drug, effective and non-invasive monitoring is needed. Liquid biopsy biomarkers, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), allow such monitoring over the course of the therapy. Interestingly, ctDNA or CTC analysis alone had less sensitivity vs. combining both, with a genotyping of 70% and 80% for CTCs and ctDNA respectively, but 100% when combined2. Vortex technology is a platform enabling label-free capture of CTCs from blood samples and genomic assays downstream3. The aim of this study is to demonstrate the sensitivity of a combined CTC and ctDNA assay through Vortex using blood samples spiked with molecularly-characterized lung cancer cell lines and then to apply this technique to matched blood and tumor samples from NSCLC patients.
Method: Lung cancer cell lines with different EGFR mutations (A549: wild type, H1975: L858R+ and T790M+, HCC827: 19del+) were used to validate our CTC workflow. Blood samples and matched tumor tissues were collected from NSCLC patients. Plasma was extracted first for ctDNA assay. CTCs were isolated from the plasma-depleted-blood using Vortex technology, immunostained (CK, Vimentin, CD45) and enumerated. DNA from CTCs, plasma and matched tumor tissue was analyzed for EGFR mutations 19del, L858R and T790M using the ctEGFR kit from EntroGen.
Results: Mutant DNA could be identified at a quantity as low as 0.5 ng (~83 cells), with a sensitivity ranging from 0.1% to 2% for a total DNA varying from 25ng (~4 CTCs among 4000 WBCs) to 1ng (~4 CTCs among 200 WBCs). We demonstrated the ability of Vortex technology to enrich CTCs from metastatic NSCLC patients. Processing of plasma-depleted-blood showed the same capture efficiency when compared to whole blood. This makes possible the detection of EGFR mutations on CTC samples collected by Vortex technology. &gt; 10 NSCLC patients are being enrolled in this study and results will be presented at AACR.
Conclusion: The ctEGFR mutation assay performed well on both Vortex-enriched CTCs and ctDNA, enabling a low cost approach to analyze EGFR mutation from a single blood tube. This non-invasive EGFR mutation analysis will be potentially a useful tool for monitoring treatment and medication guidance of NSCLC patients. 1. Calabuig-Fariñas et al. Transl Lung Cancer Res. 2016. 2. Sundaresan TK et al. Clin Cancer Res. 2016. 3. Kidess-Sigal E et al. Oncotarget 2016.
Citation Format: Haiyan E. Liu, Meghah Vuppalapaty, Clementine A. Lemaire, Charles Wilkerson, Steve C. Crouse, Jonathan W. Goldman, Elodie Sollier-Christen. EGFR mutational detection in ctDNA, Vortex-enriched CTCs and comparison to tumor tissue in non-small-cell-lung-cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1715. doi:10.1158/1538-7445.AM2017-1715