Abstract 588: Mesenchymal markers: The new avenue for circulating tumor cells detection
Authors: Ciccioli Mariacristina , Bravo-Santano Natalia , Davis Amy , Lewis Jolie , Malcolm Ross , Pailhes-Jimenez Anne-Sophie 
Published: July 2021

Abstract
Introduction: Unlike tissue biopsy, liquid biopsy allows the routine, repeat characterization of cancer at genetic, transcriptional and protein levels. Isolation of circulating tumor cells (CTCs) from a blood draw offers the potential for cancer screening, management and monitoring. Most CTC isolation systems are based on epitope-dependent CTC capture using epithelial markers. However, it is known that tumor cells can undergo epithelial-to-mesenchymal transition (EMT) when extravasating from the primary tumor to enter the bloodstream and eventually establish distant metastases. Epitope-dependent CTC detection platforms have limited sensitivity for detection of mesenchymal CTCs, leading to the inability to harvest clinically relevant CTCs for analysis, particularly in cancers with high mesenchymal phenotype such as non-small cell lung cancer (NSCLC). We developed a research use only assay for enrichment and identification of CTCs based on epithelial and mesenchymal markers using the Parsortix® system, a label-independent microfluidic device that isolates cells based on their size and compressibility.
Methods: Peripheral blood (8 – 10mL) was drawn into K2EDTA tubes from 47 metastatic breast cancer (MBC) patients and 48 metastatic NSCLC patients and processed on Parsortix systems within 36 hours after collection. Harvested CTCs were cytospun and immunofluorescently stained using an optimized panel of antibodies for detection of epithelial and mesenchymal markers. Stained slides were imaged using a BioView AllegroPlus automated imaging system.
Results: CTCs were captured from both MBC and NSCLC patients. 330 CTCs were identified from 19 MBC patients (mean: 17; range: 1-96). 58 CTCs were identified from 15 NSCLC patients (mean: 4; range: 1-22). Similarly, CTC clusters were observed in 63% (12/19) of the CTC-positive MBC patients and in 33% (5/15) of CTC-positive NSCLC patients. Cluster size ranged from 2-22 CTCs per cluster (2-17 in MBC and 2-22 in NSCLC), and the number of clusters per patient ranged from 1-13 in MBC and 1-2 in NSCLC. Phenotypically, more CTCs expressing only mesenchymal markers were harvested from NSCLC patients compared to MBC patients (38% vs 25%), with a larger proportion of CTCs expressing both epithelial and mesenchymal markers being harvested in both cancer types (59% in NSCLC and 74% in MBC). Only a small percentage of the CTCs harvested expressed just the epithelial markers.
Conclusions: This study highlights the importance of the inclusion of mesenchymal markers into CTC characterization, as the majority of the CTCs captured by the Parsortix system from MBC and NSCLC patients expressed mesenchymal markers. The assay developed in this study successfully allowed phenotyping of the harvested cells using epithelial AND mesenchymal markers, with the results suggesting that a large proportion of the CTCs harvested would have been missed with an epithelial-only based approach, particularly in NSCLC.
Citation Format: Mariacristina Ciccioli, Natalia Bravo-Santano, Amy Davis, Jolie Lewis, Ross Malcolm, Anne-Sophie Pailhes-Jimenez. Mesenchymal markers: The new avenue for circulating tumor cells detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 588.