autologous blood and marrow transplantation - Blog Science ...

514 Chapter 10: Graft Manipulation
marker and a tumor-specific marker in the same cells at the time of relapse provides
unequivocal evidence that the residual malignant cells in marrow are a source of
leukemic recurrence. In follow-up studies, we have used discriminative gene
marking with two vectors to evaluate purging techniques. A second, longer-term
purpose of these studies was to learn more about gene transfer into human
hematopoietic cells, to determine whether retroviral vectors transduce long-lived,
repopulating progenitor cells, and to ascertain whether separate populations of
hematopoietic stem cells (HSCs) could be tracked with distinguishable vectors.
PATIENTS AND METHODS
Patients
Patients diagnosed with de novo AML at St. Jude Children's Research Hospital
between 1991 and 1996 were treated on the frontline institutional study AML-91. 7
All patients received one to two courses of 2-chlordeoxyadenosine (2-CDA)
followed by one to two courses of daunomycin, cytosine arabinoside, and
etoposide. 7
All patients attaining remission who did not receive matched sibling
BMT were eligible for autoBMT. Conditioning was with busulfan 16 mg/kg and
cyclophosphamide 200 mg/kg. AutoBMT recipients were eligible to participate in
two successive marking studies approved by the Institutional Review Board,
Recombinant DNA Advisory Committee of the National Institutes of Health (NIH)
and the Food and Drug Administration (FDA). 89
During the time these gene-
marking studies were open, from September 1991 to December 1996, 44 patients
with AML in first remission received autologous transplants. Eleven were entered
on the first-generation gene marking study and 15 on the second-generation study.
Of the remaining 18 patients, three declined the gene marking component and 15
were transplanted at times when clinical grade vector was not available.
First-generation marking studies
The initial marking study in patients with AML began in September 1991 and
closed in March 1993. 10
Nucleated bone marrow (>1.5X 10 8
cells/kg body wt) was
harvested, and two-thirds were cryopreserved without manipulation. Mononuclear
cells were separated from the remaining third and transduced with either the LNL6
or GINa retroviral vector in a simple 6-hour transduction protocol in the absence
of growth factors. Both vectors encode the neo gene, which can be detected in
transduced cells either phenotypically because it confers resistance to the neomycin
analog G418, or genotypically by polymerase chain reaction (PCR). At the time of
transplant, the patient received both the transduced and unmanipulated marrow
cells. The transgene was subsequently detected in marrow progenitor cells by