Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype

Background. The epithelial-mesenchymal transition (EMT) is understood to be an important step in invasion and metastasis of cancer. It is of increasing investigational interest to identify circulating tumor cells (CTCs) that express mesenchymal markers that indicate entrance into EMT. Such cells may not express surface epithelial markers (such as EpCAM) that are often used to capture CTCs. RareCyte has developed a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF) that does not rely on surface marker capture. We developed a 5-parameter assay to identify epithelial CTCs with or without mesenchymal differentiation. Methods. Buffy coats isolated from blood by AccuCyte® separation were spread onto slides. A 5-parameter IF assay was developed with the following markers: Sytox orange (nuclear dye) / cytokeratin and EpCAM (epithelial) / vimentin (mesenchymal) / CD45, CD11b and CD105 (exclusion) / EGFR (investigational biomarker). Slides were stained with the assay on an auto-stainer, and were scanned and imaged with CyteFinder®. Specificity of the assay was validated on positive and negative control cell lines spiked into normal donor blood. It was applied to a pilot set of blood samples from 3 prostate and 3 breast cancer patients that were collected under an IRB-approved protocol. Nucleated cells that were epithelial marker-positive and exclusion marker-negative were identified as CTCs. CTCs were categorized as epithelial (epiCTC) if they were vimentin-negative and epithelial-mesenchymal (E-M) if they were vimentin-positive. In a sample from a patient with known mutation in TP53 (G244S) from tissue tumor analysis, individual epiCTCs, E-M CTCs and WBC control cells were mechanically retrieved by CytePicker® and whole genome amplification was performed. The resulting DNA was PCR amplified at the TP53 mutation site and sequenced. Results. Between 15 and 193 CTCs were counted in the patient samples. The average percentage of epiCTCs was 74% (range 33 - 100%) and of E-M CTCs was 26% (range 0 - 67%). E-M CTC fraction was higher in breast than in prostate cancer samples. EGFR-positive epiCTCs and E-M CTCs were identified. In the patient with the tumor-associated TP53 G244S mutation, 2 of 2 epiCTC, 6 of 7 E-M CTC and 0 of 6 WBC contained the mutation. Conclusions. We have developed a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype and applied it to cancer patient samples. Confirmation of malignancy of epiCTCs and E-M CTCs by single cell mutational analysis was demonstrated. The expression of EGFR on CTCs in prostate and breast cancer confirms literature reports. Citation Format: Arturo Ramirez, Nolan Ericson, Daniel Campton, Melinda Duplessis, Tanisha Mojica, Alisa Clein, Celestia Higano, VK Gadi, Daniel E. Sabath, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for identification of circulating tumor cells with epithelial-mesenchymal phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5190.