Abstract PS5-35: Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies
Authors: Sharifi Marina N , Sperger Jamie M , Gilsdorf Cole , Wolfe Serena K , Parkes Amanda , Wisinski Kari B , O'Regan Ruth M , Lang Joshua M 
Published: February 2021

Abstract
Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance.
Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-35.