Recombinant DNA Advisory Committee - 06/1-2/92 
Dr. Deisseroth explained that the presence of marked cells after a relapse will clearly 
indicate that the marrow was contaminated. The question to consider is what 
conclusions can be made if a relapse occurs and no marked cells are detected. There 
are three possible explanations for this result: (1) the marking frequency is insufficient 
in that particular patient, (2) there is a clonal evolution that would exclude marked cells, 
and (3) the bone marrow cells are not contributing to the relapse. An estimated 6,000 
blast cells will be marked in both peripheral blood and bone marrow cell preparations 
prior to reinfusion. Marking frequency of approximately 1% was observed. Therefore, 
the probability of not detecting a polyclonal relapse is below 10‘ 12 , based on statistical 
considerations. He stated confidence in detecting marked cells if relapse is occurring 
from cells that remain in the marrow used for the restoration of hematopoietic function 
by autologous transplantation. No single interpretation exists if no marked cells are 
detected. 
Dr. Deisseroth described experiments in which methyl cellulose colonies were picked and 
reverse transcriptase assays were performed. Double sequence PCR amplifications were 
performed on the cDNA obtained from these cells. Amplifications for both the 
neomycin resistance and BCR-abl genes were performed in the same tube using a 
combination of primers. A combination of primers allows for discrimination between the 
BCR-abl positive and negative leukemic cells that are either neomycin resistance positive 
or negative. Multiple primers eliminates the possibility of ambiguous results. 
Dr. Deisseroth explained that the retroviral vectors, GINa and LNL6, are distinguishable 
because GINa has an extra sequence that LNL6 does not have and GINa codes for a 
Not I site. 
In response to Dr. Brinckerhoffs question regarding the analysis of the integration sites 
with the retroviruses, Dr. Deisseroth explained that Southern blot analysis will be used 
on some patients. This procedure will determine if there has been a polyclonal evolution 
of leukemic cells attributing to relapse and whether these cells are from peripheral blood 
or bone marrow. 
Dr. Deisseroth said that the inconsistency between the two informed consent documents, 
as noted by Dr. Brinckerhoff, is that there is an original therapeutic consent document 
previously approved by the RAC and a consent form for the double marking. This 
double gene marking document is the only one to be considered by the RAC. 
Dr. Deisseroth stated that he will include a section in the informed document regarding 
a request for autopsy as requested by Ms. Meyers. 
Status Report-Dr. Deisseroth 
[702] 
Recombinant DNA Research, Volume 15 
