autologous blood and marrow transplantation - Blog Science ...

250 Chapter 6: Breast Cancer
breast cancer study. Mononuclear cells were prepared by Ficoll.
Immunocytochemical evaluation of a total of 2X10 6
mononuclear cells from the
blood or bone marrow of each patient was done. 17
The slides were air-dried
overnight, fixed for 10 minutes, and incubated for 30 minutes in a moist chamber
with a 1:20 dilution of the anticytokeratin primary antibodies AE1 and AE3 (
Signet Laboratories, Dedham, MA), followed by washing twice with Tris-HCl. As
a second step, a polyclonal rabbit anti-mouse antibody (Dako, Glostrup, Denmark)
was added, and after 30-minute incubation, the cells were washed twice with Tris-
HCl. Finally, preformed complexes of alkaline phosphatase monoclonal mouse and
anti-alkaline phosphatase (Dako, Glostrup, Denmark) were added for 30 minutes.
After two washings with Tris-HCl, the color reaction of antibody binding cells was
obtained after 10-minute incubation with 0.26% New Fuchsin solution (Aldrich
Chemical Company, Milwaukee, WI). In addition, all slides were counterstained
with hematoxylin to study cellular morphology.
The stained slides were mounted in glycerin-gelatin and examined under a light
microscope by an experienced pathologist. In an ongoing study at our hospital,
employing anticytokeratin mAbs AE1/AE3 or A45-B/B3 and immunocytochemistry,
26.6% of the bone marrow samples from 257 breast cancer patients
tested were anticytokeratin-positive. Among these samples, 5.4% of isotype
controls stained positive, suggesting that nonspecific bindings of anticytokeratin
mAbs to nonepithelial cells have taken place. Based on this experience, we always
employ morphological evaluation and negative controls using slides incubated with
isotope-matched mouse myeloma immunoglobulins followed by the APAAP
technique as described above.
RESULTS
Among the 124 patients, 29% presented with tumor cells in bone marrow and
8% in the blood at diagnosis of breast cancer (Table 1). The presence of
micrometastases in bone marrow and blood before three cycles of chemotherapy
and in the PBPC product was studied in 60 patients (Fig. 1). Despite tumorreductive
therapy with three cycles of chemotherapy, 14 of 60 patients (23%) had
tumor cells in their PBPC products. Since eight of these did not have tumor cells
detected in blood before treatment, this suggests that chemotherapy and G-CSF can
mobilize tumor cells into the blood (Fig. 1).
Six to 12 months after treatment, the patients were tested for the presence of
micrometastases in bone marrow. Of the 67 patients treated with high-dose therapy
and stem cell support, 36 had immunocytochemical examination of their marrow
6-12 months after finishing the therapy. As can be seen in Fig. 2, eleven patients
were positive in the bone marrow at diagnosis. Two had contamination of the
PBPC products and six had persistent cytokeratin-positive cells after high-dose