Detection of small numbers of
breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four
monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against
tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify
breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with
hematoxylin to confirm the identify of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human
breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of
tumor cells added and the number of
tumor cells detected was obtained over a broad range of
tumor cell concentrations. The probability of detecting
tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting
tumor at a concentration of 4
tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens,
tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing
autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained
breast cancer cells with counts ranging from 1 to 97
tumor cells per million leukocytes.
Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with
4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to
tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4
tumor cells reinfused at
autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate
breast cancer cells in hematopoietic specimens harvested for
autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of
breast cancer cells reinfused with marrow or peripheral blood stem cells.