APPENDIX C 
ABSTRACT OF PROTOCOL 
Autologous bone marrow transplantation (ABMT) is an effective way of 
treating neuroblastoma. A portion of the patient's marrow is removed during 
remission of the malignancy and cryopreserved. The patient is then given 
supralethal chemotherapy and/or radiotherapy and rescued from the associated 
marrow destruction by reinfusion of the stored cells. The two main limitations 
of the approach are: (1) the high risk of relapse, which may occur because the 
reinfused marrow is contaminated with malignant cells, and (2) the long delay 
before the marrow reconstitutes the patient with a consequent high risk of 
morbidity and mortality from infection and hemorrhage. The current proposal aims 
to transduce marker genes into harvested marrow to allow investigation of 
therapeutic strategies aimed at overcoming these problems. 
If the patients have a disease such as neuroblastoma in which malignant 
cells may contaminate the marrow, these cells may be marked by the gene transfer. 
Relapse with cells containing marker genes will then establish whether or not 
relapse originates within the patient or at least partly within the marrow cells 
harvested at the time of clinical remission. Should this latter explanation be 
correct, it would justify the procedure of marrow purging at harvest to remove 
malignant cells. At present, this procedure is of unproven benefit, but damages 
the marrow and retards engraftment, correspondingly increasing morbidity and 
mortality. Analysis of insertion sites will allow determination of clonality 
of relapse. The outcome of these investigations would affect future treatment 
strategies . 
Since marrow for ABMT is generally obtained when patients are regenerating 
their marrow after intensive chemotherapy aimed at remission induction, it is 
anticipated that a proportion of pluripotent/stem cells will be in cycle and may 
therefore be successfully transduced before cryopreservation and subsequent 
reinfusion. Marker gene insertion will be determined by the appearance of G418 
resistance amongst cultured progenitor cells in the engrafted marrow and by 
detection of vector sequences by PGR or Southern blot analysis of DNA from 
circulating cells of different lineages. We will determine what endogenous and 
exogenous stimuli, such as infection, chemotherapy, and recombinant growth 
factors, can augment the proliferation of this engrafted marrow. This 
information will help to determine whether autologous marrow genuinely re- 
populates the patient or whether it provides temporary replenishment of committed 
progenitor cells whilst surviving host stem cells gradually repopulate. In the 
future it may also be used to indicate what stimuli should be given before the 
marrow harvest to ensure maximum yield of pluripotent progenitors and which 
stimuli should be used immediately following the transplant to induce more rapid 
engraftment. With the additional knowledge obtained, better clinical protocols 
can be established. 
Recombinant DNA Research, Volume 14 
[705] 
