Performance Characteristics of a Real-Time Polymerase Chain Reaction Assay for the Detection of Epidermal Growth Factor Receptor (EGFR) Mutations in Plasma Samples of Non-Small Cell Lung Cancer (NSCLC) Patients.

Abstract

Circulating free DNA in plasma is an alternative source of tumor-derived DNA that can be a surrogate for tissue epidermal growth factor receptor (EGFR) testing. We evaluated the analytical performance of the cobas® EGFR Mutation Test v2 (cobas test), a real-time polymerase chain reaction assay designed to detect defined EGFR gene mutations in plasma from patients with advanced non-small cell lung cancer (NSCLC). We used K2-ethylenediaminetetraacetic acid plasma samples from NSCLC patients and healthy donors (HDs), along with cell line DNA. Results from a complete technical performance evaluation are described, including a comparison between NSCLC and HD plasma to support the use of surrogate samples and an independent confirmation of the limit of detection (LoD). The cobas test reported an overall percent agreement of approximately 88% for plasma samples when compared with a next-generation sequencing method. The LoD for all EGFR mutations was ≤ 100copies/mL for plasma samples. An external study confirmed the LoD for exon19 deletion, L858R, and T790M at ≤ 100copies/mL using samples derived from NSCLC patient specimens. The cobas test showed linearity between at least 50 and 10,000copies/mL for plasma samples. An internal repeatability study reported a correct call accuracy of 99.2% for plasma samples. The performance of the cobas test is equivalent when using sheared or intact cell line DNA diluted into either HD plasma or NSCLC patient plasma. The cobas test is a sensitive, robust, and accurate assay that delivers reproducible results.