Abstract B137: Development of a rapid blood-based test for EGFR sensitizing and resistance mutations in NSCLC
Authors: Foreman Trudi , Koch Kristina , Weaver Amanda , Dupuis Nicholas , Arrivo Steven , Sathyanarayana Ubaradka , Mellert Hestia , Pestano Gary 
Published: May 2016

Abstract
Approximately one quarter to one third of patients with cancer are either not candidates for biopsies, or have insufficient tissue samples gathered during their initial biopsy. These factors may limit the ability to provide a required diagnosis at the molecular level and recommend EGFR TKI therapy. The goal of this study was to develop and validate rapid, robust and highly sensitive blood-based assays that identify the EGFR sensitizing mutations L858R and exon 19 deletion (E746 - A750) in circulating DNA isolated from the plasma of patients previously diagnosed with non-small cell lung cancer (NSCLC). Additional studies were conducted to similarly detect the EGFR resistance mutation, T790M which could have implications for 3rd generation EGFR TKI therapies. The developed test is comprised of three components: 1. a whole-blood specimen collection kit that ships at ambient temperature; 2. cell free DNA (cfDNA) isolated from patient plasma samples and processed using a droplet digital PCR workflow and analyses, and 3. LIMS software for report generation. The EGFR mutation status result (classification label) is reported within 72 hours of sample shipment. Components of the assay development for the EGFR sensitizing and resistance mutation tests included: (a) ambient temperature ship stability of the sample collection kit, (b) the test system performance verification/method development studies with pre-qualified DNA standards (n = 10) and human donor samples (n = 248) and (c) sensitivity and specificity analyses with tissue. The lower limit of detection of the test system was established at 0.02% minor allele frequency. The precision (inter-day, intra-day performance) was established with validated standards as well as with human donor samples (SD &lt; 10% overall for all three assays). The robustness for all assays was evaluated over 20 consecutive days of testing, with 100% of tests passing (SD &lt; 2.0%). Sensitivity for the EGFR sensitizing and resistance mutations was shown to be 95.7% and 86.7% respectively. Specificity of the assays for their targets was established at 100% with no cfDNA in any of the healthy donors, and no cases of known tissue negative cancer donors yielding positive results to date (n = 159). In conclusion, we have developed three highly sensitive blood-based assays to identify sensitizing and resistance mutations for EGFR (L858R and exon 19 deletion E746 - A750, and T790M, respectively) in circulating DNA in plasma from patients previously diagnosed with NSCLC. These results indicate that plasma cfDNA is a reliable source for EGFR mutation analysis in clinical practice, especially for those patients unable to provide tissue-based samples. The results can be delivered to physicians within 72 hours from sample shipment and may facilitate more immediate patient management.
Citation Format: Trudi Foreman, Kristina Koch, Amanda Weaver, Nicholas Dupuis, Steven Arrivo, Ubaradka Sathyanarayana, Hestia Mellert, Gary Pestano. Development of a rapid blood-based test for EGFR sensitizing and resistance mutations in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B137.