Abstract 4534: Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations

Background: Accurate detection of driver mutations in cancer patients is vital for targeted therapy. Compared to tissue biopsy, “liquid biopsy” offers a non-invasive and more systemic approach to identify tumor mutations by assessing circulating tumor DNA (ctDNA) released from tumor cells into peripheral blood. We have developed TargetSelector TM Real-Time PCR based assays to detect low frequency mutant alleles in ctDNA. The TargetSelector TM assay uses a proprietary blocker to suppress amplification of excess WT alleles released from normal cells, while allowing specific amplification of mutants. Here we focus on five important targets: EGFR (Del19, L858, and T790), BRAF (V600), and KRAS (G12/G13), which are relevant to lung cancer, melanoma, and colorectal cancer. Methods: The TargetSelector TM assay applies a specific blocker to cover variants on a short stretch of target DNA (up to 10 bp for nucleotide variants). For example, one KRAS exon 2 blocker covers all variants on both G12 and G13 positions. The TargetSelector TM assays were first validated with cancer cell line DNA carrying mutation targets on QuantStudio 5 Real-Time PCR instruments (QS5). Sanger Sequencing was subsequently performed to confirm the mutation. Analytical validation was conducted by 3 independent operators using 5 instruments across 5 days in our CAP/CLIA certified laboratory. For ctDNA testing, whole blood samples were collected in CEE-Sure TM Blood Collection tubes and DNA extraction from plasma was performed on the QIAsymphony. Results: In total, we tested 3086 samples for EGFR, BRAF and KRAS TargetSelector TM ctDNA assays, with EGFR WT assay as the background reference. All five ctDNA assays showed >99% analytical sensitivity and >99% analytical specificity. Based on practical and theoretical estimates, each ctDNA assay demonstrated single mutant copy detection sensitivity. In the presence of 14,000 copies of WT background, the sensitivity of our ctDNA assays are: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. The inter-assay and intra-assay analyses showed r 2 >0.94, suggesting a consistent performance among operational variables. Samples tested from 20 healthy donors (100 tests in total) showed clinical specificity >99%. In the concordance study of 13 clinical samples (31 tests in total) between QS5 and ABI 7900HT platforms, TargetSelector TM ctDNA assays with the QS5 identified the same plus additional mutations compared to the 7900HT. Conclusions: TargetSelector TM ctDNA assays were validated both analytically and clinically, showing single mutant copy detection and sensitivity at 0.02% or better in a background of excess WT DNA. Implementation of the QS5 qPCR platform into our TargetSelector TM ctDNA assays leads to a higher sensitivity and faster turnaround time. These factors enable sensitive and efficient testing crucial for guiding treatment decisions and patient care. Citation Format: Shan-Fu Wu, Timothy T. Lu, Anh Pham, Jeffrey Chen, Tony Daher, Errin Samuelsz, Manisha Patel, Veena M. Singh, Lyle J. Arnold, Jason C. Poole. Validation of highly sensitive TargetSelector TM ctDNA assays for EGFR , BRAF , and KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4534.