Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAM and EpCAM CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients' blood samples both EpCAM and EpCAM cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAM cells vs. 28% of EpCAM cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAM and EpCAM CTCs. Our workflow is suitable for single CTC analysis, permitting-for the first time-assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAM and EpCAM CTCs.
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| http://dx.doi.org/10.3390/ijms18091885 | DOI Listing |
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