Recombinant DNA Advisory Committee - 09/14-15/92 
aforementioned selection procedures to purge the ABM which will be used to restore 
hematopoietic function after preparative therapy. Using the retroviral markers to 
evaluate each stage of the process, the impact of the purging will be determined. The 
use of these markers to evaluate the purging process will save both time and patients 
because the only alternative to studying the efficacy of purging is to observe the clinical 
outcome of survival and remission duration. CLL is a unique feature of this protocol. 
CLL represents a disease in which both the peripheral blood and the bone marrow are 
contaminated with abnormal cells. One patient eligibility requirement of this protocol is 
that a patient diagnosed with CLL must have an expected survival of two years without 
nonconventional therapy. 
Two events make initiation of this protocol possible. First, the chemotherapeutic 
regimens have been developed that can reduce the number of leukemia cells in the 
patient to a level that permits further purging of the marrow to render it sufficiently free 
of disease for use in the autologous setting. Transplantation alone cures 40% of CLL 
patients in the allogeneic setting. However, only a small percentage of patients are 
eligible for allogeneic transplantation because of age and donor availability. This 
protocol is designed to provide an alternative therapy option to those patients who are 
not eligible to receive allogeneic therapy. The second event that makes initiation of this 
protocol possible is that methods now exist for selecting and purging the cell population, 
namely CD34(+) selection of normal cells using the Cell Pro® column, and CD19(-) 
selection which removes abnormal cells. The two retroviruses, LNL6 and GINa, will be 
used to mark these selected populations independently. 
Conventional dose chemotherapy will be used to irradiate patients of bulk systemic 
disease and to induce remission. ABM and peripheral blood cells will be stored, 
fractionated, and transduced with the retroviral vectors. Systemic therapy will be 
administered, and the patients will be transplanted with the selected, transduced cells. 
Dr. Deisseroth presented data in which ABM cells were harvested from CLL patients 
after induction of remission by fludurabine and transduced. These ABM cells were 
transduced at a frequency of approximately 1-3%. The question of whether or not CLL 
cells are marked is not answered in this setting. In order to demonstrate CLL marking, 
ABM cells were harvested from patients who were not in remission; therefore, leukemic 
cells were predominant. Normal transduced cells were mixed with the transduced CLL 
cells at a 1:1 ratio. This mixture of transduced cells was then sorted by Fluorescence 
Activated Cell Sorting (FACS) into two populations, normal myeloid and 
CD34( + )/CD19(-), and then grown in a culture system that selectively promotes growth 
of these cells. RNA was extracted and reverse transcriptase and sequence amplification 
was performed for detection of the neo R gene. In four out of five patients, marked 
neoplastic cells were detected by PCR. Dr. Parkman asked how many cells were used 
for the PCR assay. Dr. Deisseroth answered that between 10 3 and 10 4 cells were used. 
Recombinant DNA Research, Volume 16 
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