A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer.
作者： Rita Lampignano , Liwen Yang , Martin Neumann , André Franken , Tanja Fehm
DOI: 10.3390/IJMS18091885
关键词: Circulating tumor cell 、 Flow cytometry 、 Sanger sequencing 、 Epithelial cell adhesion molecule 、 Mesenchymal stem cell 、 Amplicon 、 Metastatic breast cancer 、 Biology 、 Genome 、 Molecular biology
摘要: Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow enrich and isolate patient-matched EpCAMhigh EpCAMlow/negative CTCs within the same blood samples, investigate phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status single CTCs. We sequentially processed metastatic breast cancer (MBC) samples via CellSearch® (EpCAM-based) Parsortix™ (size-based) systems. After enrichment, captured in cassettes were stained situ for nuclei, cytokeratins, EpCAM CD45. Afterwards, sorted isolated CellCelector™ micromanipulator their genomes amplified. Lastly, PIK3CA analyzed combining an amplicon-based approach with Sanger sequencing. In 54% patients′ both identified successfully isolated. High genomic integrity observed 8% amplified vs. 28% suggesting increased apoptosis first CTC-subpopulation. Furthermore, hotspot mutations detected is suitable CTC analysis, permitting—for time—assessment heterogeneity
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