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

• Published in: Clinical cancer research 2002-05, Vol.8 (5), p.1085-1091
• Main Authors: WITZIG, Thomas E, BOSSY, Blaise, BAUER, Kenneth D, KIMLINGER, Teresa, ROCHE, Patrick C, INGLE, James N, GRANT, Clive, DONOHUE, John, SUMAN, Vera J, HARRINGTON, Douglas, TORRE-BUENO, Jose
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Breast Neoplasms - blood; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Cell Count; Gynecology. Andrology. Obstetrics; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Keratins - analysis; Mammary gland diseases; Medical sciences; Microscopy - methods; Neoplastic Cells, Circulating - chemistry; Neoplastic Cells, Circulating - pathology; Tumors
• ISSN: 1078-0432; 1557-3265

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.