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. 2024 Jan;13(1):e6843.
doi: 10.1002/cam4.6843. Epub 2023 Dec 22.

First evidence of AXL expression on circulating tumor cells in metastatic breast cancer patients: A proof-of-concept study

Thomas Bardol  1   2 , Zahra Eslami-S  1   2   3 , Doryan Masmoudi  1   2 , Marie Alexandre  4   5 , Marie Duboys de Labarre  4   5 , Angélique Bobrie  4   5 , Véronique D'Hondt  4   5 , Séverine Guiu  4   5 , Keerthi Kurma  1   2   3 , Laure Cayrefourcq  1   2   3 , William Jacot  4   5 , Catherine Alix-Panabières  1   2   3
Affiliations

First evidence of AXL expression on circulating tumor cells in metastatic breast cancer patients: A proof-of-concept study

Thomas Bardol et al. Cancer Med. 2024 Jan.

Abstract

Background: For several years, the AXL tyrosine kinase receptor, a member of the Tyro3-Axl-Mer (TAM) family, has been considered a new strategic target in oncology. AXL overexpression is common in solid tumors and is associated with poor prognosis. In this context, the detection of a subset of circulating tumor cells (CTCs) that express AXL (AXL+ CTCs) could be clinically relevant.

Methods: Immunostaining was performed to assess AXL expression in human breast cancer cell lines. The optimal conditions were established using flow cytometry. Spiking experiments were carried out to optimize the parameters of the CellSearch® system detection test. CTC enumeration and AXL expression were evaluated in patients with metastatic breast cancer (mBC) before treatment initiation.

Results: An innovative AXL+ CTC detection assay to be used with the CellSearch® system was developed. In a prospective longitudinal clinical trial, blood samples from 60 patients with untreated mBC were analyzed to detect AXL+ CTCs with this new assay. CTCs were detected in 35/60 patients (58.3%) and AXL+ CTCs were identified in 7 of these 35 patients (11.7% of all patients).

Conclusion: This newly established AXL+ CTC assay is a promising tool that can be used for liquid biopsy in future clinical trials to stratify and monitor patients with cancer receiving anti-AXL therapies.

Trial registration: ClinicalTrials.gov NCT04025541.

Keywords: AXL; circulating biomarker; circulating tumor cells; liquid biopsy; metastatic breast cancer.

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Conflict of interest statement

Catherine Alix‐Panabières is one of the patent holders (US Patent Number 16,093,934) for detecting and/or characterizing circulating tumor cells. She received an honorarium from Menarini©. William Jacot is on the Astra Zeneca, Eisai, BMS, Lilly France, Daiichi Sankyo, MSD, Novartis, Pfizer, Roche, and Seagen advisory boards and received honoraria or payment from Astra Zeneca, Eisai, BMS, Lilly France, Daiichi Sankyo, MSD, Novartis, Pfizer, Roche and Seagen. William Jacot also received meeting travel support from Astra Zeneca, Novartis, Chugai Pharma, Pfizer, Eisai, Pierre Fabre, Glaxo Smithkline, Roche, Lilly France, Sanofi, and Aventis. The remaining authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Validation of the sensitivity and specificity of the anti‐human AXL antibody FAB‐154P for the detection of AXL+ CTCs. (A) Immunocytochemical analyses. Representative images (20× magnification) of SK‐BR‐3 and MDA‐MB‐231 cells stained for AXL (red), epithelial cell adhesion molecule (EpCAM) (green) and DAPI (blue). Scale bars: 50 μm. (B) Flow cytometry analyses. Validation of the anti‐AXL antibody FAB‐154P (in blue) in SK‐BR‐3 and MDA‐MB‐231 cells compared with the isotypic control (IgG1) (in gray) and unstained cells (in red). (C) CellSearch® system analyses. SK‐BR‐3 and MDA‐MB‐231 cells added to blood samples from healthy donors were identified with the CellSearch® system using the anti‐human AXL antibody FAB‐154P in the fourth channel. All tests were performed three times and the figures depict representative results.
FIGURE 2
FIGURE 2
Representative images of AXL expression on (A) SK‐BR‐3 and (B) MDA‐MB‐231 cells using the CellSearch® system. Column 1 represents the merge picture, which is the superposition of cytokeratin (CK) expression, shown in column 2, DAPI staining, shown in column 3, and cluster of differentiation 45 (CD45) staining, shown in column 4. AXL expression is in column 5.
FIGURE 3
FIGURE 3
AXL expression analysis in CTCs from patients with metastatic breast cancer (mBC) detected with the CellSearch® system. (A) Diagram showing the number of patients without CTCs (CTCs−), with CTCs (CTCs+) and with AXL+ CTCs at baseline in the ALCINA 2 cohort (n = 60 patients). (B,C) Identification of AXL expression on CTCs using the CellTracks Analyzer II®. Column 1 shows the merge picture, which is the superposition of columns 2–4. AXL expression is shown in column 5 with an orange frame. The intensity of AXL staining varied among patients and among CTCs in the same blood sample.
See this image and copyright information in PMC

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