This question will be answered by analyzing the site where the marker gene 
has inserted using Southern blotting, and RFLP and by inverted PCR analysis 
of NB colonies (Appendix D). Where clinically indicated, biopsy specimens 
of putative relapse sites will be obtained and analyzed for neo^ gene content 
by PCR. 
6.2 Regeneration of Cryopreserved Autologous Marrow 
Patients will be monitored at weekly intervals for 6 weeks, monthly intervals 
for 6 months and annually thereafter for 5 years for the presence of cells 
containing the marker genes in the peripheral blood (PB) using FACS sorted 
populations. PB will be separated into T cells, B cells, monocytes and 
granulocytes using appropriate MAb, and each lineage examined for gene 
expression and for marker gene insertion sites by PCR and Southern blotting. 
To exclude the possibility of false positive PCR by low level contamination of 
one lineage by another, the amplification will be undertaken as quantitatively 
as possible. 
When marrow is obtained to assess engraftment and disease 
response/progression, additional cultures will analyze whether marker genes 
are present in CD34+ progenitor cells and of what lineage. 
Changes in marker gene expression in cells of different lineages will be 
correlated with changes in endogenous (serum) cytokine levels including TNF, 
ylFN, IL3, and GM-CSF (measured by commercially available immunoassays), 
induced by (e.g.) infection or chemotherapy. Ultimately changes will be 
correlated with the effects of exogenous recombinant cytokines such as IL2 
and IL3, which will be components of future submitted protocols used to 
treat these diseases. 
7.0 IMPACT OF OUTCOME ON SUBSEQUENT TREATMENT 
7.1 Source and Nature of Relapse 
There would be 2 informative outcomes if the patients relapsed. 
1) The majority of relapse patients have no marked cells. However, 
occasional patients relapse with cells containing the marker gene and 
this relapse is monoclonal. This would imply the patients receiving 
marrow harvested in remission are in fact receiving occasional 
malignant "stem cells" and that relapse is monoclonal in nature. 
Detection of this event would be of low probability (see Section 8 - 
below). 
Recombinant DNA Research, Volume 14 
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