Abstract 1955: Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic breast cancer
Authors: Sharifi Marina N. , Helzer Kyle T. , Sperger Jamie M. , Bootsma Matthew L. , Krause Hannah , Gilsdorf Cole S. , Wolfe Serena K. , Kauffman Zachary , Tevaarwerk Amye J. , Burkard Mark E. , Parkes Amanda M. , O'Regan Ruth M. , Wisinski Kari B. , Zhao Shuang G. , Lang Joshua M. 
Published: June 2022

Abstract
Background: Phosphatidylinositol-3-kinase (PI3K) activating mutations are found in 30-40% of breast cancers, and the PI3K inhibitor alpelisib is FDA-approved for PIK3CA-mutated hormone-receptor positive metastatic breast cancer (MBC). However, rates of intrinsic alpelisib resistance are as high as 40%, and there is an ongoing need for novel biomarkers to help guide patient selection. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) may better reflect the heterogeneity of metastatic disease than tissue biopsy, and are amenable to longitudinal analysis. By combining targeted ctDNA sequencing with CTC transcriptional profiling and quantitation of PI3K pathway phospho-proteins, we have developed the first multiplex assay to simultaneously assess genomic alterations and CTC PI3K pathway activity via liquid biopsy.
Methods: Peripheral blood was collected from MBC patients with known PIK3CA mutation status serially prior to starting new standard therapies and on treatment. Cell-free DNA was extracted from plasma and sequenced using a custom capture panel (IDT). CTCs were isolated with VERSA microfluidic technology integrating capture with downstream analysis. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or staining on chip for phospho-AKT pS473 and total AKT or phospho-rpS6 pS235/S236 and total rpS6, followed by quantification of single cell phospho/total protein mean fluorescence ratios.
Results: In a pilot cohort of 14 patients, mean CTC phospho-AKT/total AKT protein expression ratio was higher in patients with PIK3CA mutations compared to patients without mutations (0.37 vs 0.26, p&lt;0.01). A transcriptional signature of PI3K activity was also higher in CTCs from patients with PIK3CA mutations than from patients without. Among patients with PIK3CA mutations, there was inter-patient heterogeneity in CTC phospho-AKT and phospho-rpS6, consistent with variability in pathway activation. One patient with a tissue PIK3CA mutation 5 years prior did not have the mutation detected in ctDNA at time of liquid biopsy, and had low phospho-AKT and transcriptional PI3K activity in CTCs. In serial sampling of a patient receiving a PI3K inhibitor, CTC phospho-AKT was decreased on treatment, and increased at time of progression.
Conclusion: We have demonstrated the feasibility of a comprehensive liquid biopsy for simultaneous monitoring of PI3K pathway mutations in ctDNA and PI3K pathway signaling in CTCs via transcriptional profiling and phospho-protein expression in patients with MBC. Future work will prospectively evaluate this assay in patients receiving alpelisib for PIK3CA-mutated MBC. This has the potential to complement PIK3CA mutation status as a biomarker of sensitivity to PI3K therapies, and may also provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs on treatment.
Citation Format: Marina N. Sharifi, Kyle T. Helzer, Jamie M. Sperger, Matthew L. Bootsma, Hannah Krause, Cole S. Gilsdorf, Serena K. Wolfe, Zachary Kauffman, Amye J. Tevaarwerk, Mark E. Burkard, Amanda M. Parkes, Ruth M. O'Regan, Kari B. Wisinski, Shuang G. Zhao, Joshua M. Lang. Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic breast cancer [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 1955.