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Detection of Circulating Cytokeratin-positive Cells in the Blood of Breast Cancer Patients Using Immunomagnetic Enrichment and Digital Microscopy

Department of Laboratory Medicine and Pathology [T. E. W., T. K., P. C. R.], Division of Medical Oncology [J. N. I.], Department of Surgery [C. G., J. D.], Section of Biostatistics [V. J. S.], Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, and ChromaVision Medical Systems, Inc., San Juan Capistrano, California 92690 [B. B., D. H., J. T-B., K. D. B.]

Purpose: To examine the feasibility for identifying and enumerating cytokeratin positive (CK+) cells in the peripheral blood of breast cancer patients.

Experimental Design: Blood specimens from 34 normal donors (negative controls), 15 samples to which carcinoma cells were added (positive controls), and 84 breast cancer patients [27 node-negative (N-), 29 node-positive (N+), and 28 metastatic] were studied. RBCs were lysed with ammonium chloride and the resulting cell suspension incubated with anti-EpCAM-conjugated immunomagnetic beads for carcinoma cell enrichment. Immunomagnetically selected cells were placed on slides; stained for CKs 8, 18, and 19; and evaluated with an automated digital microscopy system that rapidly scanned the slide and collected images of cells meeting predefined staining and cytomorphological criteria. A montage of the CK+ cells was reviewed to confirm tumor cell morphology.

Results: Eighteen specimens (9 normal, 2 N-, 4 N+, and 3 metastatic) were excluded because of poor cytomorphology or staining artifact. All 15 of the positive controls [95% confidence interval (CI), 78–100%] and none of the 25 negative controls (95% CI, 0–14%) demonstrated CK+ cells. Twenty-one of the 75 (28%; 95% CI, 18–40%) samples from breast cancer patients demonstrated CK+ cells including 76% of patients with metastatic disease (95% CI, 55–91%), 8% with N+ disease (95% CI, 1–26%), and none of those with N- disease (95% CI, 0–14). The mean number of CK+ cells detected in the 21 CK+ patients was 18.4 (range, 1–120).

Conclusions: Breast carcinoma cells can be detected in the blood from a significant fraction of metastatic breast cancer patients using immunomagnetic cell enrichment and digital microscopy. The incidence of CK+ cells was low in those with resected N+ disease (at most 26%) and those with resected N- breast cancer (at most 14%). This technique could be used in large prospective studies of patients with breast cancer to learn whether the detection of rare carcinoma cells is a useful predictive or prognostic factor.

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