Abstract P1-05-35: Identification of Circulating Tumor Cells captured by the FDA Cleared Parsortix® PC1 System from the Peripheral Blood of Metastatic Breast Cancer Patients using Immunofluorescence and Cytopathological Evaluations
Authors: Ciccioli Mariacristina , Moore Richard , Kim Kyu Kwang , Khazan Negar , Miller Michael C. , Pailhes-Jimenez Anne-Sophie 
Published: March 2023

Abstract
Background: Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a non-invasive surrogate source of tumor material to investigate tumor characteristics in real-time. The Parsortix® PC1 System, the first FDA-cleared medical device for the capture and harvest of CTCs from peripheral blood of metastatic breast cancer (MBC) patients for use in subsequent user-validated downstream analyses, enables the epitope independent capture of CTCs with diverse phenotypes based on cell size and deformability. In this study, CTCs isolated from the blood of MBC patients by the Parsortix® PC1 System were identified using an immunofluorescence (IF) based assay for detection of epithelial CTCs followed by Wright-Giemsa staining and cytomorphological review. The aim of this study was to determine the proportion of MBC patients and self-declared female healthy volunteers (HVs) that had one or more CTCs identified in the population of cells harvested from their peripheral blood by the Parsortix® PC1 System. Methods: Peripheral blood from 75 HVs and 77 patients with MBC was prospectively collected into K2EDTA tubes at the University of Rochester Medical Center. The blood collected from each subject (8.6±1.2mL) was processed on a Parsortix® PC1 System within 8 hours of collection. The cells harvested by the system were cytospun onto a charged slide and IF stained using an optimised antibody panel. The IF panel consisted of a nuclear dye (DAPI), positive selection markers targeting epithelial CTCs (Cytokeratins (CK) and EpCAM), and negative selection markers targeting white blood cells, such as lymphocytes, macrophages, granulocytes, monocytes, fibroblasts, and cells of megakaryoblastic potential. The stained slides were imaged using fluorescence microscopy and CTCs were defined as nucleated cells (DAPI+) that were positive for CK and/or EpCAM and negative for the blood lineage markers. The IF slides were subsequently stained with Wright-Giemsa and analysed by a qualified pathologist using light microscopy. Morphological features of malignant cells were used to define and identify CTCs. All laboratory testing and analysis was performed by operators blinded to the clinical status of each subject. Results: On the evaluable IF-stained slides, cells classified as CTCs based on their IF staining pattern were identified in 45.3% (34/75) of the MBC patients (range = 0 – 125 CTCs, mean = 7) and in 5.6% (4/71) of the HVs (range = 0 – 8 CTCs, mean = 0). No EpCAM+, CK- CTCs were identified in either MBC patients or HVs. In the 34 MBC patients with one or more CTCs observed, 70.6% had only CK+, EpCAM- cells, while the remaining 29.4% had at least one CK+, EpCAM+ cell. In the HVs, one out of the four CTC-positive donors had only CK+, EpCAM+ cells while the other three had only CK+, EPCAM- cells. On the evaluable Wright-Giemsa stained slides, cells classified as CTCs by the pathologist were identified in 57.1% (40/70) of the MBC patients (range = 0 – 41 CTCs, mean = 4) and in 4.4% (3/68) of the HVs (range = 0 – 14, mean = 0). Conclusions: This study demonstrated the ability of ANGLE’s Parsortix® PC1 System to capture and harvest CTCs from a significantly larger proportion of MBC patients compared to HV subjects. The presence of epithelial cells in subjects without diagnosed disease has been previously described in the literature, with their significance being unclear. This study also demonstrated that the cells harvested by the Parsortix® PC1 System can be successfully evaluated using both IF staining and Wright-Giemsa cytomorphological assessment for the identification of CTCs. Interestingly, a high proportion of the identified CTCs did not express EpCAM, further highlighting the limitations of using EpCAM-based approaches to capture CTCs.
Citation Format: Mariacristina Ciccioli, Richard Moore, Kyu Kwang Kim, Negar Khazan, Michael C. Miller, Anne-Sophie Pailhes-Jimenez. Identification of Circulating Tumor Cells captured by the FDA Cleared Parsortix® PC1 System from the Peripheral Blood of Metastatic Breast Cancer Patients using Immunofluorescence and Cytopathological Evaluations [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-05-35.