Detection of circulating cytokeratin-positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy.
OBJECTIVE
To examine the feasibility for identifying and enumerating cytokeratin positive (CK+) cells in the peripheral blood of breast cancer patients.


METHODS
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.
Witzig
Thomas E
TE
Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA. witzig@mayo.edu
Bossy
Blaise
B
Kimlinger
Teresa
T
Roche
Patrick C
PC
Ingle
James N
JN
Grant
Clive
C
Donohue
John
J
Suman
Vera J
VJ
Harrington
Douglas
D
Torre-Bueno
Jose
J
Bauer
Kenneth D
KD
D001943:Breast Neoplasms / Q000097:blood* / Q000378:metabolism / Q000473:pathology; D002452:Cell Count; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis*; D008853:Microscopy / Q000379:methods; D009360:Neoplastic Cells, Circulating / Q000737:chemistry* / Q000473:pathology
D007633:Keratins