population. One would predict that the number of cells with high MDR-1 
would increase with each cycle of therapy. The nadir and duration of 
the nadir at each dose level will be compared with the percentage of 
cells which are retroviral MDR-1 positive. This will test if the 
degree to which the marrow is replaced with MDR-1 positive cells will 
determine the resistance to Taxol. 
RT PCR will be conducted on cells collected after transduction to 
obtain a population average for the level of expression of the 
retroviral MDR-1. RT PCR will also be conducted on a colony-by-colony 
basis using methylcellulose for this analysis. Cells will be taken from 
the peripheral blood and marrow, cultured in Dexter culture, and then 
plated in methylcellulose, or plated directly in methylcellulose. This 
will be repeated after each cycle of Taxol, and following recovery of 
marrow function following the autologous transplant. 
We will also distinguish between the MDR-1 which arises from the 
endogenous genes, and from the retroviral transgenome. There will be 
two 5' primers: one which is homologous to sequences in the 5' 
untranslated region of the endogenous MDR- 1 mRNA, which are not 
present in the 5' untranslated residual viral sequences which appear in 
the MDR-1 primary transcript. The 3 ' primer will originate from DNA 
within the first exon of the MDR-1 transcription unit. Thus, the length 
of the product and the type of DNA in each primer product will permit 
discrimination of the endogenous and the retroviral gene product. Thus, 
we will be able to identify the level of expression of the endogenous 
and exogenous MDR-1 throughout the exposure to Taxol. 
In order to determine if the introduction of MDR-1 into the cells of 
the marrow has slowed down the rate of recovery, we will compare the 
rate of recovery of the white blood cell count seen in autologous bone 
marrow transplant patients when advanced breast or other cancer 
patients are given the thiotepa, cyclophosphamide preparative regimen 
outlined in our protocol, when given an engrafting dose of CD34 
selected autologous hematopoietic cells. We and others have now had 
experience with CD34 selected cells as reconstituting populations 
following intensive systemic preparative regimens, which are identical 
to the one we will use in this MDR-1 study. The median day to 500 and 
1000 PMN/cu mm for CD34 transplants in heavily pretreated epithelial 
neoplasm patients is 17 days (range 10-22) . If a delay in hematopoietic 
recovery occurs following transplant with the MDR modified marrow, we 
will be able to detect this if three patients or more exhibit a delay 
of recovery to greater than 40 days to a PMN count of 500/mm^. 
Schema for Treatment Plan: 
I. Non-ablative dose chemotherapy (cyclophosphamide, 4 gms/m^) to 
recruit early precursor cells into a proliferative phase and to 
mobilize them into the peripheral blood. 
II. A total of 4 X 10® nucleated cells/kg of peripheral blood cells 
for breast cancer patients and an engrafting dose of 20,000 
CFUGM/kg will be collected. One half of it will be concentrated 
by the COBE pheresis instrument CD34 selected, and transduced as 
described in Appendix D. There will be at least a total of one 
million CD34 peripheral blood stem per kilogram of body weight 
for the transplant which will be composed of modified and 
unmodified cells. 
Recombinant DNA Research, Volume 19 
[533] 
