2) Most relapses contain both marked cells and unmarked cells and the 
insertion site may be polyclonal. This would imply that marrow 
contains a multiplicity of cells capable of inducing relapse. 
These different outcomes would have different clinical implications. 
Outcomes 1 and 2 would imply that purging is indeed a worthwhile procedure 
and should be further explored. 
Outcome 2, in which relapse is polyclonal and a high proportion of patients 
relapsing therefore show a positive marker, would also provide a unique 
method for assessing the effectiveness of different techniques of purging. Such 
methodology is now entirely lacking (see paragraph 1.4), 
7.2 Stem Cell Marking 
Analysis of the stimuli required in vivo to induce stem cells into cycle, and to 
induce proliferation of cells of different lineages would greatly simplify efforts 
to grow adequate marrow in vitro from small aliquots for successful 
subsequent ABMT. Moreover an indication of the cytokines responsible for 
stem cell cycling would help in planning more effective chemotherapy and in 
optimizing the timing of marrow harvest for subsequent storage and re- 
infusion. 
8.0 STUDY SIZE AND STATISTICAL CONSIDERATIONS 
8.1 Transduction of Malignant Progenitor Cells 
The transduction efficiency for fresh malignant neuroblastoma cell lines is 
between 0.3% and 3% of clonogeneic cells assessed on colony assays in G418 
and by PCR (Appendbf A). At present, there is no way of assessing whether 
"clonogenic cells" and neuroblastoma "stem cells" are identical or have the 
same transduction efficiency. If we assume that transduction efficiency for 
clonogenic cells and stem cells is identical and take a figure of 1% as average 
efficiency, then only 0.3% of the total malignant "stem cells" present in a 
harvested marrow will be transduced in the 30% aliquot exposed to the 
vector. This makes it unlikely that marrow relapse from <10 progenitors 
would be detected unless >50 patients were studied - a number not currently 
feasible. Polyclonal relapse (possibility 2 in section 7.2 above) would be more 
readily detected with a probability that would increase as the number of NB 
"stem cells" present increased. The marrow relapse rate in patients receiving 
ABMT for stage D neuroblastoma is >60% at 12 months. A polyclonal 
relapse would therefore be detectable in a cohort of 12 individuals with a 
[ 686 ] 
Recombinant DNA Research, Volume 14 
