Relationship between the detection rate of EpCAM-positive circulating tumor cells (CTCs) in metastatic breast cancer patients and the CTC enrichment technology.

Abstract

e21066 Background: Immunomagnetic EpCAM-based methods are successfully used to enrich and enumerate circulating tumor cells (CTCs) in metastatic breast cancer patients (mBC pat). In vitro data raised concerns whether EpCAM dependent CTC assays may enrich EpCAM negative (EpCAM-) CTCs. Methods: Tumor cells were identified with the anticytokeratin antibody, A45-B/B3. (i) We evaluated the EpCAM expression of 3 cell lines MCF-7, ZR-75-1 and Hs587T with flow cytometry. (ii) An anti-EpCAM immunomagnetic enrichment method, MACS HEA MicroBeads (MACS) was compared to a non-anti-EpCAM based new density centrifugation method, OncoQuick plus (OQ+), using spiking experiments. (iii) To validate these data in vivo, peripheral blood from 26 mBC pat was collected to compare the two methods. (iv) To explore EpCAM expression of the respective patients' CTCs, blood was enriched with the OQ+ procedure. An aliquot of enriched blood was immunocytochemically stained with a fluorescence-labeled EpCAM antibody. The other aliquot was used to verify EpCAM expression with RT-qPCR. Results: (i) MCF-7 and ZR-75-1 cell lines showed a strong (>90%), Hs587T tumor cells had a low (< 5%) or no EpCAM expression, respectively. (ii) MACS recovered significantly more of a definite number of spiked tumor cells than OQ+ in EpCAM positive (EpCAM+) cell lines (p = 0.002 and p = 0.007, respectively). OQ+ recovered significantly more EpCAM- Hs587T cells (p < 0.0001). (iii) With MACS 42.3% of mBC pat. had CTCs detected (median 0). With OQ+ CTCs were spotted in 69.2% of pat (median 8). The positivity rate of detected CTCs did not differ significantly between MACS and OQ+ (p = 0.065). A significantly higher number of CTCs was enumerated with OQ+ (p = 0.018). (iv) Irrespectively of the EpCAM detection method, 19% of mBC pat had EpCAM+ CTCs. MACS detected significantly (p = 0.001) more EpCAM+ CTCs. No association was found between CTCs detected by OQ+ and their EpCAM expression. Conclusions: The CTC enrichment method should be chosen according to the respective CTCs' EpCAM expression. Further investigations have to determine whether the accuracy of prognostication of mBC pat depends on the detection of EpCAM± CTC subpopulations. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca