Pre-IDE Submission: Clinical Protocol 
Neuroblastoma Bone Marrow Purging System 
BAXTER HEALTHCARE CORPORATION, HYLAND DIVISION 
described herein. We will rely on these extensive previous data as well as preclinical 
studies in model systems for establishment of appropriate antibody concentrations and 
bead/cell ratios for the purging procedure. Thus, safety and dosing studies for the use 
of magnetic microspheres and monoclonal antibodies in the clinical trials will not be 
addressed here. Our studies are intended to (1) determine whether gene marking is a 
safe and appropriate tool to evaluate efficacy of purging and, if so, (2) to use gene 
marking to provide information about the source of relapse, and the efficacy of purging 
in neuroblastoma patients undergoing autologous bone marrow transplantation. The 
detailed objectives and methods of analysis are discussed further below. 
The following trial will use gene marking of tumor cells to monitor the contribution of 
reinfused bone marrow (purged and unpurged) to relapse in neuroblastoma patients. The 
proposal is to transduce neomycin resistance marker genes, contained in two closely 
related but distinguishable vectors, into two aliquots of marrow obtained for ABMT. 
One aliquot of marked marrow will be immunomagnetically purged using the Baxter 
Neuroblastoma Bone Marrow Purging System. This will be mixed with the second 
marked, unpurged aliquot and reinfused into the patient. 
Recent reports have demonstrated that immunocytological analysis is a highly sensitive 
technique for the detection of neuroblastoma infiltration of bone marrow. This assay, 
in combination with a newly developed assay for clonogenic neuroblastoma cells, will 
be used to quantitate tumor contamination pre- and post-purging, and at relapse. 
Clonogenic tumor cells can then be cultured, their identity confirmed by 
immunoperoxidase staining, and the presence of the marker genes detected by PCR 
analysis (Part 3 of Appendix C). The number of marked progenitor cells contributing 
to the relapse will be estimated and the ratio of malignant cells marked with each vector 
will be calculated. As each patient would act as their own control, analysis should be 
independent of other variables, such as chemotherapy regimen. 
This combination of assays will therefore provide (i) quantitative data on the removal of 
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Recombinant DNA Research, Volume 16 
