Human Gene Therapy Subcommittee - 11/30/90 
marking, transduction of various differentiated cell lines, and the clonality of 
leukemia in relapse. 
Dr. Epstein asked about the ability to perform a clone-by-clone analysis using 
inverse PCR. Dr. Ihle said there are different techniques for performing clonal 
analysis, but the method they employed uses a restriction enzyme to cut the LTR 
region of a leukemia virus. The ligated segment is then allowed to circularize, or is 
inserted into a plasmid, and is then cut again at a known restriction site. PCR is 
used to amplify the fragment, and then the size and characteristics of the integration 
site are analyzed by Southern blot. 
Dr. Mclvor asked what morphological differences will be looked for in the AML 
blast colonies. Dr. Ihle explained that the colonies could be analyzed by cytospin, 
histochemistry, and cytogenetic marker studies, and that normal CFU-C can easily be 
differentiated from AML blast colonies. 
Dr. Mclvor asked about the ability to fractionate normal and AML cells before 
performing molecular analysis. Dr. Ihle said that what is important in this regard is 
knowledge of one of the transforming events. The EVI-1 gene is used because it is 
not normally expressed at detectable levels in normal bone marrow and is present in 
AML bone marrow. Recently, investigators have identified myeloid transforming 
genes which aids in the identification of leukemia by looking for alpha-retinoic acid 
receptor activation. This receptor is a definitive marker of leukemia with a 15/17 
translocation, as well as the KEN gene which has been identified as residing at 
another translocation break point in 5-10% of leukemias. 
Dr. Epstein asked how much heterogeneity was found in relapsed marrow. Dr. Ihle 
said that in the case of AML, the cytogenetic markers are the same in most cases. 
Dr. Mclvor summarized by saying that it appeared that there was some finite risk 
which is not assessable at present. There is still not enough detail provided on how 
many cells are involved and how much virus is involved in terms of assessing the 
efficiency of transduction of the marrow prior to reinfusion. Further, the details of 
the PCR analyses need to be more clearly defined in writing as to morphology and 
negative results. A feasibility study needs to be undertaken in an animal model to 
assure that the virus can be used to label marrow and tumor cells, and that this 
marking experiment can be evaluated by the techniques proposed in the protocol. 
Dr. Ihle said that unless he was told exactly what cell line to use, one that would 
have properties similar enough to spontaneous AMLs, it would be impossible to 
perform such a study because of the heterogeneity that exists between AMLs. 
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Recombinant DNA Research, Volume 14 
