6.0 EVALUATION OF EFFECTS 
6. 1 Relapse 
If relapse occurs, then the malignant blasts will be separated by FACS and it 
will be determined whether or not the relapse cells contains either marker 
gene. By using the appropriate primers, the size of the PCR fragment 
amplified will indicate whether either or both marker genes are present in the 
bulk population. Semi-quantitative PCR will indicate the relative proportions 
of each marker gene. Individual clones of leukemic cells will be grown in 
methylcellulose colonies, and assigned to the LNL6 or GIN aliquot (and 
hence to one or the other purging methods) on the basis of PCR fragment 
size. 
6.2 Regeneration of Cryopreserved Autologous Marrow 
When marrow is obtained to assess engraftment and disease progression, 
additional cultures will analyze which marker genes are present in progenitor 
cells and in what lineages. Patients will also 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 monoclonal antibodies 
(MAb), and each lineage examined for the proviruses. Again, clonogenic 
analyses of progenitor cells grown in methylcellulose will be used to compare 
the contribution of each marrow aliquot to reconstitution. 
7.0 INFERENCES WHICH MAY BE DRAWN FROM STUDY RESULTS 
7. 1 Source of Relapse 
There are at least three informative results of the study. 
1) Children relapse with marked cells and both vectors are equally 
represented. 
This result would confirm that purging is necessary but would suggest 
that the current techniques are inadequate. Studies of new purging 
methods would be necessary. 
2) Children relapse and the cells are only marked with the vector 
associated with one of the two purging methods. 
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