Although molecular assays exist for the neoplastic 
lymphoid cells in the CLL population (PCR for T cell 
rearrangement and immunoglobulin gene rearrangement in 
the B cells of the CLL population) , so that the level of 
CLL cells left in the marrow population can be studied, 
no formal assays exist to determine if the neoplastic 
early progenitor cells, which have reconstituting 
capability, have been removed. In addition, at the time 
of relapse, it is impossible to tell if the relapse 
arises from the neoplastic cells which are left following 
systemic preparative therapy, or if the relapse occurs 
from leukemia cells left in the marrow infused into the 
patient following in vitro purging of the marrow and 
delivery of intensive combination chemotherapy and TBI. 
In order to be able to identify the origin of relapse: 
residual hematopioetic cells in the marrow after purging, 
versus the relapse from disease left in the systemic 
circulation following intensive preparative therapy, we 
are proposing to use safety modified retroviruses to 
follow the efficiency of purging with the CD34 positive 
selection of the normal myeloid stem cells and the 
negative selection of the CLL cells with negative 
selection mediated by CD19 negative selection. 
In the current protocol, we will mark 30% of the marrow 
cells following the CD34 positive selection with the LNL6 
retrovirus, and then mark the marrow following the CD19 
negative selection with the GINa virus. We will follow 
the marking protocol outlined in Appendix G. This will 
involve the culture of marrow cells from CLL ptients on 
autologous stromal feeder layers in the presence of cell 
free supernatants of producer cell lines for safety 
modified retroviruses. We will rinse the cells. A small 
aliquot will be incubated in non-selective conditions for 
48 hours, and then added as an innoculum of cells to 
semi-solid culture medium, colonies grown up under 
selection by G418, and then RNA extracted from single 
colonies which are picked 10-14 days following the 
initial innoculation . The major part of the marrow will 
be frozen for transplantation. 
The mRNA can be reverse transcribed, and the cDNA split 
into two halves and amplified independently. One 
combination can be amplified with primers for the 
rearranged immunoglobulin molecule, and the neo gene of 
either the LNL-6 or GINa virus. Another half of the cDNA 
will be amplified with the actin and the immunoglobulin 
molecule primers. The DNA of the LNL6 and GINa virus can 
be distinguished by the PCR assay. 
With the help of these PCR assays, we can characterize 
the population of the cells at relapse as to whether the 
relapse occurred due to cells left after the CD34 
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Recombinant DNA Research, Volume 16 
