provirus, apheresis will be performed to study the MDR-1 transduced populations. 
Row cytometry studies using the MRK-16 antibody and/or rhodamine efflux will be 
done on cells collected by apheresis to study Pgp expression and function. 
Furthermore, these cells will be sorted by FACS and the various subpopulations tested 
for the MDR-1 provirus by PCR and in situ hybridization. Hematopoietic clonogenic 
assays will be grown with and without the addition of taxol. 
8.15 Serum for wild type virus and Western blot analysis will be obtained every 3 months. 
8.16 Upon the death of a patient, an autopsy will be requested and multiple organ sites will 
be analyzed by PCR for the presence of the MDR-1 gene. 
9.0 Statistical Considerations 
The main objectives of this pilot trial are to study the feasibility' of obtaining bone marrow 
engraftment with hematopoietic stem cells bearing the MDR-1 provirus after high dose 
chemotherapy and to study whether the transduced cells can be positively selected by post- 
ABMT chemotherapy. The trafficking and time course patterns of the MDR-1 marked cells in 
the peripheral blood and bone marrow will be studied by serial sampling of these sites during 
and after bone marrow engraftment. Patients with residual or progressive breast cancer post- 
ABMT will be treated with taxol or vinblastine therapy plus G-CSF. The dose of taxol or 
vinblastine will be increased by 10% each cycle to achieve a neutrophil nadir < 1000/mm 3 
(Section 5.10). When this nadir count has been achieved, the dose of taxol or vinblastine w ill 
be held constant for successive cycles unless dose reductions for toxicity are needed (Section 
7.0). Blood and bone marrow samples will be obtained to determine if positive selection for 
the MDR-1 transduced hematopoietic cells has occurred with taxol or vinblastine therapy. 
Three blood samples obtained before and after chemotherapy will be analyzed in triplicate by 
quantitative PCR for the percentage of cells containing proviral MDR-1. (The PCR 
methodology and the procedure for determining copy number are outlined in Appendix I.) 
The null hypothesis for this pilot, feasibility study is that taxol or vinblastine therapy will not 
increase the number of circulating and bone marrow’ hematopoietic cells that express the 
transduced MDR-1 gene. Each patient will act as her own control with regard to the 
percentage of cells carrying proviral MDR-1 pre- and post-chemotherapy. 
The table below shows the statistical power of the assay. Although our assay has a measured 
standard deviation of 0.28 of the mean (Appendix I), the table below' assumes a value of 0.3. 
The first column is the factor by which the treatment actually increases the percentage of 
circulating MDR-1 -transduced cells, and the second column is the probability of finding a 
significant difference. In an individual patient, if any single cycle of taxol (vinblastine) 
treatment resulted in a three fold increase in proviral copy number, our assay w'ould detect a 
difference significant at the two-tailed p=0.05 level in 92 percent of the cycles. We anticipate 
treating 18 patients, and each patient with residual disease or relapse will likely receive several 
cycles of salvage chemotherapy. 
Factor 
Power 
fSD=0.3f 
2.0 
57.0% 
2.5 
80.0% 
3.0 
92.0% 
3.5 
97.0% 
4.0 
99.0% 
4.5 
99.7% 
5.0 
99.9% 
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