A Novel Workflow to Enrich and Isolate Patient-Matched EpCAM(high) and EpCAM(low/negative) CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer

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(high) and EpCAM(low/negative) 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((R)) (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(high) and EpCAM(low/negative) cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAM(low/negative) cells vs. 28% of EpCAM(high) cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAM(high) and EpCAM(low/negative) CTCs. Our workflow is suitable for single CTC analysis, permittingfor the first timeassessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAM(high) and EpCAM(low/negative) CTCs.