Liquid biopsies have the potential to revolutionize the way physicians select personalized anti-cancer therapies, monitor patient responses to treatment, and characterize acquired resistance to cancer drugs. New tests that use a simple peripheral blood draw offer snapshots of a patient‘s total tumor DNA mutation profile and are attractive because of their minimally-invasive modality and because they integrate information from both primary and metastatic disease. Currently, most plasma cell-free DNA (cfDNA) mutation detection tests used in clinical research detect known hotspot mutations in a limited number of genes. Technologies that interrogate multi-gene panels in cfDNA are advancing, but commercially-available options suitable for clinical use are limited, come at a high cost, and are not customizable. We designed and developed a customized, next generation sequencing-based, liquid biopsy assay capable of detecting somatic mutations in 87 breast cancer genes involved in cell cycle and estrogen receptor signaling. Targeted regions (147 Kb) were enriched using hybrid capture resulting in an average capture specificity and uniformity of 65.93% and 96.38%, respectively. When tested on cfDNA from healthy donors (n=14), we demonstrated a level of specificity >99.99%. Analytical sensitivity of 0.1% was established on HapMap and reference mutant cell line DNA. Using a pool of HapMap genomic DNA (n=10), 96% (48/50) of SNPs with expected allele frequency of 0.1% were detected. In reference mutant cell line DNA with 1% or 0.1% mutant allele frequencies, we were able to reliably detect all mutations present at 1% and mutations at 0.1% in 50% of the cases. Assay validation on plasma cfDNA with matching tumor from ER+, HER2- breast cancer patients will be presented. In conclusion, we developed a highly sensitive and specific liquid biopsy assay to interrogate 87 breast cancer-related genes. The high level of specificity and sensitivity makes the test ideal not only for detecting known cancer gene hotspot mutations but also for detection of novel gene mutations that may arise during treatment as a result of acquired drug resistance.

Citation Format: Maruja E. Lira, Tao Xie, Shibing Deng, Jennifer Kinong, Jingjin Gao, Zhou Zhu, Nathan Lee, Paul Rejto, Jadwiga Bienkowska, James Hardwick, Kai Wang, Stephen Huang. Liquid biopsy testing allows highly-sensitive detection of plasma cfDNA mutations in 87 breast cancer-related genes [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 2749. doi:10.1158/1538-7445.AM2017-2749