Abstract 534: An ultrasensitive high-plex assay detecting 24 PIK3CA mutations using SAGAsafe® technology and 6-color Crystal Digital PCR™

Abstract

Abstract Starting from a minimally invasive liquid biopsy sample such as a blood sample, one can determine genetic and biological properties of cancer by measuring circulating tumor DNA (ctDNA). ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. The focus of this study was to demonstrate the performance of the patented SAGAsafe digital PCR chemistry using 6-color Crystal Digital PCR, by adapting a SAGAsafe assay for a clinically relevant oncogene, PIK3CA. PIK3CA encodes the p110-alpha kinase and is the target of a recently approved drug, alpelisib, for metastatic breast cancer. By combining the power of the SAGAsafe technology with that of 6-color Crystal Digital PCR on the naica system®, we present a highly multiplexed, ultrasensitive proof-of-concept PIK3CA detection assay capable of detecting and quantifying 24 PIK3CA mutations in a single digital PCR reaction. By evaluating the sensitivity, specificity and precision of the high-plex 6-color PIK3CA digital PCR assay, we show that despite the complexity of the assay, the data were extremely clean resulting in highly accurate quantifications down to mutant allele frequencies (MAFs) in the 0.01% range. In addition, superb PIK3CA mutation detection specificity and sensitivity were observed with Limits of Blank ranging from 0.003% to <0.001% MAF, and Limits of Detection ranging from 0.009% to 0.002% MAF in a high background of wild-type DNA. Moreover, a high concordance was observed between Crystal Digital PCR PIK3CA results and NGS data on breast cancer biopsies. By joining 6-color Crystal Digital PCR and ultrasensitive SAGAsafe technology, high sensitivity and specificity can be maintained even in a complex multiplex digital PCR assay. This proof-of-concept assay sets the stage for the future development of ultrasensitive, highly multiplexed cancer detection panels to achieve better patient stratification and monitoring during therapy. Citation Format: Miguel Alcaide, Anthony M. George, Yilun Chen, Cécile Jovelet, Sahbi Ben Kilani, Lao H. Saal, Allison C. Mallory. An ultrasensitive high-plex assay detecting 24 PIK3CA mutations using SAGAsafe® technology and 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 534.