Abstract 2300: Validation of highly sensitive ctDNA assays for ESR1 resistance mutations with the NGS TargetSelectorTM enrichment technology
Authors: Wu Shan-Fu , Arnold Lyle J. , Shrivastava Jolly , Poole Jason C. 
Published: August 2019

Abstract
Background: Acquired resistance to anti-hormonal therapy is a common reason for treatment failure in advanced and metastatic breast cancer (mBRC). Mutations in the ESR1 gene, encoding the estrogen receptor (ER) protein, are often associated with this drug resistance. Accurate and timely ESR1 mutation detection may enable early identification of progression and prediction of treatment failure. In contrast to using difficult and painful tissue biopsies, “liquid biopsy” offers a non-invasive and systemic approach to identify solid tumor mutations in peripheral blood by assessing circulating tumor DNA (ctDNA). Biocept has developed the highly sensitive TargetSelectorTM NGS assay to detect low frequency mutant alleles in ctDNA using a patented blocker that suppresses excess WT alleles, while still allowing amplification of tumor derived mutant sequences. Here, we focus on a five-codon ESR1 mutation hotspot that imparts tumor cell resistance to anti-hormonal therapy in mBRC.
Methods: The TargetSelectorTM assay applies a specific blocker on a short stretch of target DNA (up to 13 bp). The ESR1 assay consists of two blockers in the region of codons 534-538 of the ESR1 gene. We validated the TargetSelectorTM assays by spiking low level g-block DNA fragments (IDT Inc.) carrying point mutations for each of the targeted ESR1 sites, into human placental DNA. Mutant sequences were enriched via PCR and mutations were subsequently confirmed on the Illumina MiSeq down to a limit of detection (LOD) of 0.01-0.05% minor allele frequency (MAF). For ctDNA testing, whole blood was collected in Biocept CEE-SureTM Blood Collection tubes, and total nucleic acid extraction from plasma was performed on the QIAsymphony.
Results: In total, we tested &gt;600 samples for ESR1 TargetSelectorTM ctDNA assays, with ESR1 WT as the background reference. The assay shows &gt;99% analytical sensitivity (at 0.05% MAF) and &gt;97% analytical specificity. Based on practical and theoretical estimates and using ddPCR as an orthogonal verification method, the ESR1assays demonstrate single mutant copy detection for most tested positions, and in all cases showed an LOD of at least 0.05% (5 mutant copies in a background of 10,000 WT copies). Samples tested from 20 healthy donors (40 tests in total) showed high clinical specificity, indicating the test is competent for use in a CLIA certified, CAP accredited laboratory setting.
Conclusions: Biocept’s TargetSelectorTM ctDNA assays are validated both analytically and clinically, showing single mutant copy detection and LOD at 0.05% or better in a background of excess WT DNA. Implementation of the Illumina MiSeq platform into our TargetSelectorTM assays leads to higher sample throughput, greater sensitivity and a faster turnaround time than Sanger sequencing. These factors enable a sensitive and efficient test crucial for guiding treatment decisions and patient care.
Citation Format: Shan-Fu Wu, Lyle J. Arnold, Jolly Shrivastava, Jason C. Poole. Validation of highly sensitive ctDNA assays for ESR1 resistance mutations with the NGS TargetSelectorTM enrichment technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2300.