The Detection of Isolated Tumor Cells in Bone Marrow Comparing Bright-Field Immunocytochemistry and Multicolor Immunofluorescence
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The detection of isolated tumor cells in bone marrow by immunocytochemistry (ICC) has been reported to predict progression of early-stage breast cancer. The most common staining procedure uses bright-field ICC with cytokeratin (CK) antibodies to label isolated tumor cells. However, this method can result in false-positive staining events. We used multicolor immunofluorescence (IF) to develop a more specific assay for detecting isolated tumor cells in marrow samples from breast cancer patients.
We compared ICC and IF side by side for detection of cancer cells and false-positive staining events on bone marrow aspirates from breast cancer patients, bone marrow from healthy donors, and healthy donor blood spiked with cancer cells. The primary target for isolated tumor cell detection was CK for both methods. IF used an additional set of antibodies to label hematopoietic cells (HCs).
The detection rate of CK+ events in breast cancer patient bone marrow aspirates was 18 (58%) of 31 for ICC and 21 (68%) of 31 for IF. However, with IF, 17 of 21 CK+ cases were stained with HC markers and thus were identified as false-positive events. A surprisingly high CK+ event rate was observed in healthy donor blood and marrow. In all healthy donor samples, CK+ events were readily identified as HCs by IF. Detection sensitivity of spiked cancer cells in donor blood was similar for both methods.
There is a high frequency of CK+ events in blood and marrow, and it is important to note that this is observed both in patients with and those without cancer. IF with multiple HC markers allows straightforward discrimination between CK+ cells of hematopoietic and nonhematopoietic origin.
KeywordsImmunocytochemistry Bone marrow Cytokeratin Immunofluorescence
The authors thank Elaine Cahoon, Patricia Lutton, Jennifer Spano, and Jenne Wax at the University of Vermont, Vermont Cancer Center, and Augusta Kosowicz of the Charleston Area Medical Center for Cancer Research for their assistance in protocol development, patient recruitment, and sample delivery. We also thank Julie Malloy for administrative assistance and Joseph Tessitore for obtaining material from the primary breast tumors at Fletcher Allen Health Care. Supported by The University of Vermont General Clinical Research Center (GCRC MO1 RR00109), the National Institute of Health (PHS CA74137-06S1), the Charleston Area Medical Center Foundation and Charleston Area Medical Center Institute, Charleston, WV.
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