Enrichment of paired EpCAMhigh and EpCAMlow/negative CTCs within metastatic breast cancer blood samples and mutational analysis of the oncogene PIK3CA
20 September 2018 (online)
Circulating tumour cells (CTCs) are mainly enriched with EpCAM-based approaches (i.e. CellSearch® [CS]) which may overlook EpCAMlow/negative cells with an epithelial-mesenchymal plasticity. Besides, the mutation status of PIK3CA has so far not been analysed in EpCAMlow/negative CTCs.
We aimed to establish and validate a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs from metastasized breast cancer (MBC) patients and to characterize the PIK3CA mutation status in single CTCs.
CTC-enrichment procedure was established using spiked blood samples. They were processed with CellSearchTM followed by size-selective Parsortix™ (6.5 µm) and immunostained in situ. CTCs were harvested and detected by epifluorescence microscopy. Single cell isolation was performed with CellCelector™ micromanipulator. 52 MBC blood samples were processed according to the workflow and genomic DNA was amplified from single CTCs (Ampli1™ WGA). PIK3CA hotspot regions in exons 9 and 20 were amplified and resulting amplicons were Sanger sequenced.
The average recovery rate of MCF-7 BC cells with Parsortix™ was 33 ± 4%. In 54% of MBC blood samples with EpCAMhigh CTCs, EpCAMlow/negative cells could be detected. Positivity rates did not correlate. High quality WGA products were observed in 22.5 ± 18.4% EpCAMhigh CTCs vs. 7.6 ± 10.2% EpCAMlow/negative CTCs. CTCs harboring different PIK3CA hotspot mutations were detected in 4/10 patients.
We provide a robust workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs for single cell downstream analysis. For the first time, we show heterogeneous PIK3CA mutation status in EpCAMlow/negative CTCs.