autologous blood and marrow transplantation - Blog Science ...

522 Chapter 10: Graft Manipulation
low numbers of tumor cells in hematopoietic tissues. 7-9
Reported numbers of
micrometastatic tumor cells range from an average of 5-10 tumor cells per 10 5
to
10 6
hematopoietic cells in bone marrow to 1 tumor cell per 10 5
to 10 6
in autoSCT
grafts. 9-11
Therefore, MRD detection assays should have a detection sensitivity of
at least 1 tumor cell in 10 6
. MRD detection assays must also be highly specific.
False-positive results due to nonspecific crossreactivity, coexpressed antigens,
and/or user variability severely limit the clinical value and applicability of any
tumor detection assay. 4-9
In addition, the assay should be of limited complexity, so
that its ease of use will enable widespread use in research and clinical settings.
Several investigators have explored the use of immunologic or immunomagnetic
cell capture systems in an effort to improve tumor detection
sensitivity. 14-17
While these various systems have met with success, some
procedures are labor-intensive and others may be limited in application by
disruption of cell viability and integrity. A tumor enrichment technology that
maintains cell integrity and is rapid and simple to perform would likely assist
transplanters to measure occult tumor cells in autoSCT grafts more efficiently and
more accurately.
Thus, the sensitive detection of hematogenous micrometastases in the
HDC/autoSCT treatment setting may be a powerful prognostic tool in selecting
those patients who are tumor-negative and may have a better clinical outcome after
HDC/autoSCT. In the autologous graft engineering area, the enhanced detection of
hematogenous micrometastases may also assist transplant physicians in selecting
autoSCT products that are tumor-free or identifying those grafts that are tumorcontaminated
and may require additional tumor cell purging. We report on the
development of a laboratory-scale tumor enrichment column that incorporates
avidin-biotin cell selection technology for the enrichment of tumor cells from
blood, marrow, and stem cell apheresis products.
MATERIALS AND METHODS
Tumor cell seeding experiments
Breast cancer cell line CAMA was seeded into normal donor bone marrow
(BM) and peripheral blood progenitor cell (PBSC) specimens at concentrations
ranging from 1:10 3
to 1:10 8
. A pre-TEC portion of each seeded sample was
reserved for immunocytochemical (ICC) staining, and the remaining sample was
processed on the TEC as follows. The seeded samples were incubated with an antiepithelial-cell
capture antibody (NeoRx, Seattle, WA) for 30 minutes at room
temperature, then washed with 1% bovine serum albumin (BSA). Each sample was
resuspended in up to 3 mL of 5% BSA and processed on the TEC. The flowthrough
(FT) fraction (i.e., nonenriched cells) was collected and reserved, and the