Detection of Breast Cancer Cells in Blood Using Immunomagnetic Bead Selection and Reverse Transcription-Polymerase Chain Reaction.

Background: A sensitive and specific method for the detection of occult breast cancer cells may prove clinically useful. The purpose of this study was to investi gate cytokeratin-19 (CK-19) and gross cystic disease fluid protein (GCDFP) as potential reverse transcription-polymerase chain reaction (RT-PCR) targets for the detection of breast micrometastases. Through positive selection of breast epithelial cells by immunomagnetic bead separation, two RT-PCR assays were developed. Methods and Results: Positive selection of breast epithelial cells was performed using Ber-EP4 monoclonal antibody bound to magnetic beads. RNA was isolated and RT-PCR performed using CK-19 and GCDFP as targets. Detection sensitivity was five T47D cells spiked into 10 mL of normal blood for either target. In all, 100% (3 9/39) of normal bloods were negative for CK-19, whereas only 87% (34/39) were negative with GCDFP. In 15 patients with metastatic disease including 3 without treatment and 12 on either tamoxifen or chemotherapy, CK-19 was positive in 67% (10/15) of patients and GCDFP yielded positives in 27% (4/15) of patients tested. Conclusions: The detection of CK-19 and GCDFP in bloods from patients with metastatic breast cancer may be beneficial in determining the course of therapy, as well as having potential prognostic and diagnostic applications. Although CK-19 appears to be the more sensitive and specific marker, further investigation with both targets is warranted.