Abstract 539: Development of a novel 5-plex copy number ddPCR assay for ctDNA analysis
Authors: Wiggins Jennifer M. , Syeda Mahrukh , Polsky David 
Published: June 2022

Abstract
Background: Analysis of cell-free circulating tumor DNA (ctDNA) from blood provides an opportunity to detect potential mechanisms of resistance to targeted therapies in melanoma patients. Approximately 20% and 17% of patients undergoing BRAF targeted therapy develop resistance associated with copy number increases in either BRAF or cylinD1 (CCND1), respectively. Detection of BRAF or CCND1 copy number gains prior to radiographic tumor progression could potentially prompt a therapeutic switch aimed at treating patients with smaller tumors burdens, which may improve survival. Conventional droplet digital PCR (ddPCR) for copy number analysis uses two probes, one target and one reference gene (2-plex). To improve the accuracy and sensitivity of the copy number analysis and account for possible genetic alterations in the reference gene, we performed a 5-probe multiplex (5-plex) reaction using three reference genes and two targets.
Methods: We used cell lines with known copy number gains for the BRAF gene (M395-R) and CCND1 gene (SkMel-187) to identify copy number increases using ddPCR. To estimate the limit of detection of the copy number gains in plasma, high and low concentrations of the cell line DNAs were spiked into healthy plasma and divided into three replicate samples of 3ml each. DNA was extracted from plasma using the Circulating DSP NA Kit (Qiagen). The ddPCR reaction mix used 4X Supermix and five different copy number specific probes targeting: two genes of interest, BRAF (7q34) and CCND1 (11q13.3); and three control genes: FOXI3 (2p11.2), AGO1 (1p34.3) and TTC5 (14q11.2). Each probe was titrated to display a unique cluster location on the 2D plot. Probe concentrations were: BRAF: FAM 2X; CCND1: FAM 1X; FOXI3: HEX 2X; AGO1: HEX 1X; TTC5: FAM 0.7X/HEX 0.4X. Each extraction was divided into 4 replicate wells. Following droplet generation, gene copies were analyzed using the QX Manager 1.2 Standard Edition for advanced analysis.
Results: Probe titration resulted in 32 quantifiably distinct ddPCR clusters on a single 2D plot. Copies/ul of the reference genes were used as a benchmark for diploid status. We determined that the M395-R cell line had 45 copies of the BRAF gene and that SKMel-187 had eight copies of the CCND1 gene compared to diploid PBMC DNA (Promega). Based on the spike-in experiments, we estimate that we can detect copy number gains in plasmas with 10% or 20% tumor fraction when tumor cells have approximately 45 or 8 copies of the target gene, respectively.
Conclusion: We successfully multiplexed five copy number assays in a single ddPCR reaction, and showed that copy number changes could be identifiable with as little as 10% tumor fraction within plasma when the tumor-associated copy number gains were relatively high. Overall, the 5-plex assay has the potential to be excellent tool for copy number detection in blood, and can be customized as needed for other targets.
Citation Format: Jennifer M. Wiggins, Mahrukh Syeda, David Polsky. Development of a novel 5-plex copy number ddPCR assay for ctDNA analysis [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 539.