Introduction: Isolation of circulating tumor cells (CTCs) is widely recognised as a prognostic biomarker in metastatic breast cancer. Recently, it was shown that CTC clearance following specific therapies may predict chemosensitivity in triple negative breast cancer (TNBC) patients. However, a limited amount of CTC research is available in TNBC due to the inability of epitope-dependent CTC platforms to capture CTCs in a sufficient proportion of patients, especially when evaluating non-metastatic patients (Stage I-III). ANGLE has developed a Research Use Only (RUO) workflow including the Parsortix® system, an epitope-independent microfluidic device that isolates and harvests rare cells from blood based on their size and deformability, followed by an immunofluorescent (IF) assay for the identification and characterization of CTCs. This five-color multiplex IF assay enables the identification of both epithelial and mesenchymal CTCs as well as the investigation of other targets of interest on the CTCs.

Methods: 7.5mL of peripheral blood drawn into a Streck CellFree DNA Blood Collection Tube from 11 TNBC patients (four Stage IV and seven Stage IIIa) was processed on Parsortix® system between 96 and 144 hours post collection. Harvested CTCs were cytospun onto a charged slide and immunofluorescently stained using an optimised antibody panel allowing for detection of epithelial and mesenchymal CTCs. Stained slides were imaged using a BioView Allegro Plus automated imaging system.

Results: CTCs were identified in 9 (82%) of the 11 TNBC patients, with a median of 6 and mean of 30 CTCs identified per patient. CTC clusters were harvested by the Parsortix® system and were observed in the majority (8/9) of the CTC-positive patients. Cluster size ranged from 2-68 CTCs per cluster, and the number of clusters per patient ranged from 1-50. Phenotypically, a large proportion of the CTCs identified expressed only mesenchymal markers (80%), with the remainder expressing both epithelial and mesenchymal markers (20%). In 6 of the 7 non-metastatic (Stage IIIa) patients, high numbers of CTC clusters were detected. Lower numbers of CTC clusters were detected in 3 (75%) of the 4 metastatic (Stage IV) patients.

Conclusions: This study demonstrated the ability of ANGLE’s multiplex IF assay to detect and phenotype CTCs. The proportion of non-metastatic TNBC patients found to have CTCs was much higher compared to previous reports using epitope-dependent systems (82% vs 25%). This highlights the advantages of epitope-independent CTC capture in TNBC. ANGLE’s RUO Parsortix® system and multiplex IF assay provide an efficient solution for the harvesting and characterization of multiple CTC phenotypes, with the added advantage of being able to process the blood between 96 and 144 hours post collection, allowing for the shipment of samples at room temperature for centralised analysis in support of global clinical trials.

Citation Format: Amina Mezni, Matthew Hardy, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Epithelial and mesenchymal CTC detection in triple negative breast cancer patients using ANGLE’s Parsortix®system [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 969.