TY - JOUR
T1 - Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support
AU - Franklin, Wilbur A.
AU - Shpall, Elizabeth J.
AU - Archer, Philip
AU - Johnston, Charles S.
AU - Garza-Williams, Sara
AU - Hami, Lisa
AU - Bitter, Mitchell A.
AU - Bast, Robert C.
AU - Jones, Roy B.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1996
Y1 - 1996
N2 - 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; BrE-3 ] 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 identity 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-hydroxyperoxycyclophosphamide (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.
AB - 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; BrE-3 ] 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 identity 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-hydroxyperoxycyclophosphamide (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.
KW - Bone marrow
KW - Breast neoplasms
KW - Immunohistochemistry
KW - Monoclonal antibodies
KW - Transplantation
UR - http://www.scopus.com/inward/record.url?scp=0029823584&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029823584&partnerID=8YFLogxK
U2 - 10.1007/BF01807031
DO - 10.1007/BF01807031
M3 - Article
C2 - 8932871
AN - SCOPUS:0029823584
SN - 0167-6806
VL - 41
SP - 1
EP - 13
JO - Breast Cancer Research and Treatment
JF - Breast Cancer Research and Treatment
IS - 1
ER -