C. HESDORFFER 4/93 
250/ig/m 2 /day of GM-CSF until the white cell count exceeds 1,000 
as is standard procedure. Harvested autologous peripheral blood 
stem cells will also be given as support if necessary. 
Both peripheral blood and marrow of all patients transduced with 
the MDR gene will be tested for the presence of MDR DNA and RNA 
by PCR and Southern blot analyses. In addition, we will use FACS 
sorting with the 17F9 antibody (32) to determine the percent of 
transduced cells in which the MDR gene is expressed. We will 
determine the cell populations (granulocytes or lymphocytes) 
containing and expressing the MDR gene in peripheral blood and in 
marrow samples using FACS sorting with antibodies specific for 
these populations as well as gating on the basis of size and 
morphology (32) . Aliquots for analysis of CFU-GEMM and 
BFU-E colonies will also be collected and colonies resistant to 
colchicine measured. Individual colonies will be analyzed for 
transduction by MDR PCR. 
We will use the outpatient facilities of the General Clinical 
Research Center for follow-up visits for patients treated at 
CPMC . Marrow will be obtained from patients at 3-6 month 
intervals for six months posttransplantation and then at six- 
month intervals thereafter. We will draw blood monthly after 
transplantation to determine the presence and expression of the 
MDR gene. At each visit, we will examine patients for 
progression of their underlying malignancy and for other 
manifestations potentially resulting from the retrovirus. 
Safety tests will be done following human gene transfer into bone 
marrow. Transduced bone marrow cells from all patients will be 
cocultured with Mus-dunni cells and supernatants assayed for the 
presence of reverse transcriptase and in the S+L assay. Serum 
from MDR-transduced patients will be plated on untransduced 3T3 
cells and colchicine resistance of these cells tested 
(16,17,32,47). In addition, the presence or absence of the MDR 
gene by PCR will be analyzed in the recipient 3T3 cells after 
exposure to the serum. Serum from patients as well as transduced 
cells will also be assayed periodically by co-culture with mus 
dunni cells and S+L assays. 
A potential theoretical risk in this protocol is that undetected 
residual cancer cells in the patient's marrow will be transduced 
with the MDR gene and become resistant to MDR-responsive drugs 
such as taxol . This is more often true for breast cancer than 
for ovarian or brain cancer. It is possible that these rare 
cells could become the predominant tumor population and lead to 
disease relapse and death. While these events might all occur, 
we do not believe they represent a significant risk to these 
patients. First, the number of residual cells in the marrow will 
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