autologous blood and marrow transplantation - Blog Science ...

260 Chapter 6: Breast Cancer
Table 1. Flow cytometry on cytokeratin--stained aspirates versus routine pathology
Flow cytometry Pathology
50% positive (35/70) 17% positive (12/70)
12 positive samples 12 positive pathology
23 positive samples 23 negative pathology
Maximum sensitivity: Maximum sensitivity:
1 tumor cell/3.OX 10 5
cells 1 tumor cell/7.0X10 3
cells
Flow cytometry allowed analysis of 10 5
-10 6
cells, resulting in an assay that
detected the presence of epithelial cells three times more often than pathology. All
specimens with morphologically confirmed metastases were also positive using our
approach, lending credence to the accuracy of cytokeratin +
events identified with
flow cytometry. Aspirates determined positive by both methods had the highest
level of tumor contamination, with an estimated frequency of at least 1 tumor cell
in 7X10 3
nucleated cells. Flow cytometric analysis of immunofluorescently stained
cells was 40 times more sensitive than pathology and could detect as few as 1
tumor cell in 3X10 5
cells.
Tumor contamination of mobilized peripheral stem cell collections (PSCC) has
been associated with overt disease in the marrow. 71718
We found that the presence
of tumor cells with a frequency as low as 1 in 10 6
cells still predicted tumor
contamination in PSCC from stage III and IV breast cancer patients (Table 2).
Despite the inclusion of cytoreductive therapy in the mobilizing regimen, 89% of
the patients with cytokeratin +
marrow had detectable tumor cells in one to two
leukapheresis products. Designating a negative marrow as prerequisite to apheresis
reduced the risk of tumor presence in the stem cell product. Of the 70 patients with
cytokeratin-negative marrow, 91% also had negative leukaphereses.
Initial development and adaptation of the staining procedure from cell lines to
primary tissue was dependent on the availability of positive samples. Bone marrow
aspirates collected in EDTA anticoagulant could be stored in aliquots and studied
for 2-3 days because tumor cells remained detectable in the unprocessed portions.
Conversely, whole blood samples, which contained cytokeratin +
cells on the day of
collection, had no detectable cells 24 hours later. This observation raised concerns
Table 2. Cytokeratin status of marrow vs. peripheral stem cell collections: 79 stage III
and IV breast cancer patients
Aspirate results Tumor frequency — apheresis + apheresis
70 negative patients 0 64(91%) 6 (9%)
9 positive patients 1/1.2X10 5
(1/9X10 3
-1/10 6
)
1 (11%) 8 (89%)