Purpose: Circulating tumor cells (CTCs) have a tremendous potential for diagnosis and treatment of breast cancer patients. Here, we performed a unique analysis of all atypical circulating cells isolated with a filtration-based technology from metastatic breast cancer (mBC) patients. Patients and methods: The PERMED-01 study enrolled patients with mBC, refractory to systemic therapy, and with an accessible lesion to biopsy. We analyzed atypical circulating cells isolated from patients' blood at

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... he time of inclusion using Screencell® Cyto device. For 23 out of 91 analyzed patients, this was completed by advanced immunofluorescence staining of atypical circulating cells. Subsets cut-offs were established using a two-component Gaussian finite Mixture Model, and evaluated for correlation with clinico-pathological data, including progression-free survival (PFS) and overall survival (OS). Results: Three subsets of atypical circulating cells, absent from controls (n=7), were observed in cancer patients (n=91): isolated (iCTCs), Clusters (CTM), and Giant CTCs (gCTCs). CTCs' median number was 8.33 per mL. Co-expression of stem and drug resistance markers was associated with intermediate epithelial to mesenchymal transition phenotype in CTM and gCTCs, but not in iCTCs. Presence of gCTC was associated with shorter PFS and OS. Concerning PFS, assigning an immunofluorescence-based Epithelial to Mesenchymal status improved their prognostic value. Conclusion: This study brings to light the diversity of CTCs in mBC patients and their specific molecular profiles regarding epithelial to mesenchymal transition, stemness and drug resistance status. It also highlights the involvement of an atypical circulating cell subset, the gCTCs, as a prognostic factor for PFS and OS.