autologous blood and marrow transplantation - Blog Science ...

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clonogenic assays in the presence or absence of G418, 11
or in marrow colonies or
peripheral blood cells by PCR. 11
Second-generation marking studies
Second-generation studies that used double discriminative gene marking to
compare two different purging techniques 9
enrolled patients between September
1993 and November 1996. The two vectors used in these studies, GIN and LNL6,
differ in 3' noncoding sequence, so primers can be designed that will result in
amplification of fragments of different sizes. In this protocol, at least 10 8
marrow
cells/kg were frozen without manipulation as a safety backup. The remaining
marrow mononuclear cells were divided in half and randomized to marking with
GINa or LNL6. The aliquots were then randomly assigned to the two purging
techniques under evaluation. We initially compared the pharmacologic "gold
standard" hydroperoxycyclophosphamide (4HC) with an immunologic purge by
culture with interleukin (IL)-2 12
(in collaboration with Dr. H. Klingemann). Later,
we used CD15 antibodies 13
(provided by Dr. E. Ball) instead of IL-2. Patients
received both purged aliquots at transplant and the unmanipulated fraction if
engraftment was delayed. In patients who relapsed, marrow and peripheral blood
specimens were evaluated for presence of both marker genes.
RESULTS
First-generation gene marking studies
Twelve patients were enrolled (11 with AML in first remission and one with
AML in second remission), and follow-up now ranges from 68 to 84 months. Four
patients have relapsed. In three of these patients, malignant cells identified by a
tumor-specific marker have contained the marker gene. The fourth patient was
uninformative, as his blasts did not have a leukemia-specific marker, so it was not
possible to determine if the PCR signal was arising from normal or malignant cells.
These data show definitively that marrow harvested from patients with AML in
apparent remission may contain residual leukemic cells with the potential to
contribute to a subsequent relapse. These observations suggest that effective
purging of marrow might remove this source of leukemic cells and potentially
improve the outcome of autoBMT.
These studies also allowed assessment of the efficiency of gene transfer to
normal hematopoietic cells. Four patients in this cohort relapsed and two died (one
sepsis, one myocarditis), but the remaining six have now been followed for 68-84
months. The marker gene has continued to be detected in these patients for up to 7
years in both clonogenic assays performed on marrow cells and PCR assays