Comprehensive Longitudinal Linear Mixed Modeling of CTCs Illuminates the Role of Trop2, EpCAM, and CD45 in CTC Clustering and Metastasis

Background/Objectives: Breast cancer is the most commonly diagnosed cancer worldwide, with high rates of distant metastasis. While circulating tumor cells (CTCs) are the disseminatory units of metastasis and are indicative of a poor prognosis, CTC heterogeneity within individual patients, among breast cancer subtypes, and between primary and metastatic tumors within a patient obscures the relationship between CTCs and disease progression. EpCAM, its homolog Trop2, and a pan-Cytokeratin marker were evaluated to determine their contributions to CTC presence and clustering over the study period. We conducted a systematic longitudinal analysis of 51 breast cancer patients during the course of their treatment to deepen our understanding of CTC contributions to breast cancer progression. Methods: 272 total blood samples from 51 metastatic breast cancer (mBC) patients were included in the study. Patients received diverse treatment schedules based on discretion of the practicing oncologist. Patients were monitored from July 2020 to March 2023, with blood samples collected at scheduled care appointments. Nucleated cells were isolated, imaged, and analyzed using Rarecyte® technology, and statistical analysis was performed in R using the lmerTest and lme4 packages, as well as in Graphpad Prism version 10.4.1. Results: Both classical CTCs (DAPI+, EpCAM+, CK+, CD45– cells) and Trop2+ CTCs were detected in the blood of breast cancer patients. A high degree of correlation was found between CTC biomarkers, and CTC expression of EpCAM, Trop2, and the presence of CD45+ cells, all predicted cluster size, while Pan-CK did not. Furthermore, while analyses of biomarkers by receptor status revealed no significant differences among HR+, HER2+, and TNBC patients, longitudinal analysis found evidence for discrete trajectories of EpCAM, Trop2, and clustering between HR+ and HER2+ cancers after diagnosis of metastasis. Conclusions: Correlation and longitudinal analysis revealed that EpCAM+, Trop2+, and CD45+ cells were predictive of CTC cluster presence and size, and highlighted distinct trajectories of biomarker change over time between HR+ and HER2+ cancers following metastatic diagnosis.