Enrichment, isolation and PIK3CA mutational analysis within patient's matched EpCAMlow/negative and EpCAMhigh CTCs in metastatic breast cancer
09 May 2017 (online)
Circulating tumor cells (CTCs) are epithelial cells found in the peripheral blood stream of cancer patients. CTCs are mostly detected by EpCAM-based systems which miss cells with an EpCAMlow -mesenchymal phenotype.
Our aim was to isolate and characterize the PIK3CA hotspots mutations in patient's matched EpCAMlow/negative and EpCAMhigh-CTCs in metastatic breast cancer.
First, we developed a robust enrichment-isolation workflow by combining the CellSearch®, the size-selective Parsortix™ and the CellCelector™ micromanipulator. Then, 58 blood samples were processed as mentioned. Enriched cells were stained for nuclei, cytokeratin, EpCAM and CD45, detected by fluorescence microscopy and isolated. Genomic DNA was amplified (WGA). One high quality WGA product of an EpCAMlow-cell was used for array-based comparative genomic hybridization (aCGH) to confirm its malignant origin. All WGAs exhibiting phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) exons 9 and 20 amplicons were subsequently Sanger sequenced.
We detected both EpCAMlow/negative and EpCAMhigh-cells in 55% of blood samples without any correlation in positivity rate. High genomic integrity was observed in 16% of 145 EpCAMlow/negative-cells vs. 59% of 107 EpCAMhigh-cells from 13 patients. The aCGH profile exhibited several genomic aberrations providing hinds of malignancy for the EpCAMlow/negative subgroup. CTCs harboring the mutated and the wildtype PIK3CA were detected in 4/10 patients. Two of them showed mutations in the EpCAMhigh subgroup only, one in the EpCAMlow/negative subgroup only and one in both CTCs subgroups.
Concluding, our workflow is suitable to isolate the EpCAMlow/negative-CTCs and to show, for the first time, the heterogeneous PIK3CA status within patient's matched EpCAMlow/negative and EpCAMhigh-CTCs.