A novel ESR1 mutation assay for single circulating tumor cell by applicaion of DEPArray (TM) System and Digital Droplet PCR

Abstract Introduction: Although circulating tumor cells (CTC) display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. ESR1 mutations are a major mechanism of acquired endocrine resistance in metastatic breast cancer (MBC). We previously reported that ESR1 mutations in circulating tumor DNA (ctDNA) was associated with worse prognosis in MBC (2020 ASCO). Herein, we report a new ultra-high sensitivity approach of ESR1 mutations assay for single CTC by using DEPArray system and Droplet Digital PCR (ddPCR). Methods: Whole blood sample (7.5ml/each) was collected from stage IV MBC patients and then CTC enumeration was performed in CellSearch™ System by targeting the EpCAM antigen (2020 AACR #3120). The single CTC and single white blood cell (WBC) were then isolated from CellSearch cartridges by using DEPArray࣪ System (Menarini). The single CTC DNA was isolated by QIAamp DNA Micro Kit (Qiagen). ESR1 mutations hotspots including p.E380Q, p.Y537S and p.D538G were analyzed by using QX200™ ddPCR System (Annealing/Extension=58 °C, 40 cycles) and the corresponding probes (Mutation-FAM and Wild type-HEX mixed) for p.E380Q-dHsaMDS500014536, p.Y537-dHsaMDS975379796, p.D538G- dHsaMDS460485301 respectively (Bio-Rad). Meanwhile, corresponding plasma ctDNA was analyzed by Guardant 360 Health NGS-based assay for a 73 genes panel for single nucleotide variants. Results: Positive controls (using 0.00000325ng pure positive DNA) and negative control (using water) were performed in each ddPCR assay. In cohort 1: total of 8 samples including 4 single CTC samples and 4 single WBC samples were collected using the DEPArray system. Wild type ESR1 (p.E380Q, p.Y537S and p.D538G) was detected in all 4 single CTC samples and 4 single WBC samples. There were 3 copies, 3.4 copies, 1.6 copies and 1.8 copies of p.E380Q mutation droplets found in 4 single CTC samples when there were no p.Y537S or p.D538G mutations found in these samples. There was no ESR1 mutation found in the corresponding ctDNA. In cohort 2: Two samples including 4 CTCs and 100 CTCs were collected from CellSearch cartridges. There were 0.09 vs 1 copies (8.25%) and 0.1 vs 0.31 copies (24.39%) of p.E380Q mutation droplets found in these two samples respectively, compared to ctDNA results of 1.80% and 0 respectively. Furthermore, there was 0.08 vs 0.67 copies (10.66%) of p.D538G mutation droplets were found in the sample including 100 CTCs compared to 12.70% mutations detected in the corresponding ctDNA. Conclusion: Our new approach verified the feasibility of ESR1 mutations assay in very low amount DNA from single CTCs with high sensitivity even when ESR1 mutation could be found in CTCs but not ctDNA. This approach may offer a strong evidence for early disease metastasis, efficacy of individualized treatment monitoring and personalizing cancer therapy for precision medicine. Citation Format: Qiang Zhang, Paolo D'Amico, Marwa Manai, Carolina Reduzzi, Andrew A. Davis, Lorenzo Gerratana, Weijun Qin, Jianhua Jiao, Saya L. Jacob, Jeannine Donahue, Youbin Zhang, Lisa Flaum, Massimo Cristofanilli, Leonidas C. Platanias, Ami N. Shah, William Gradishar. A novel ESR1 mutation assay for single circulating tumor cell by application of DEPArray࣪ System and Digital Droplet 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 1950.