NEW ORLEANSPositive/negative purging of peripheral blood stem
cell (PBSC) grafts allows preparation of highly purified CD34+
fractions and up to 6 log of tumor cell depletion in patients with
chronic lymphocytic leukemia (CLL), according to a study conducted at
the University of Kiel, Germany. Peter Dreger, MD, reported the
results at the 41st annual meeting of the American Society
of Hematology (ASH).
Peripheral blood stem cells are often heavily contaminated with
leukemic cells, making effective purging strategies mandatory, Dr.
Dreger said. Although tumor cell contamination can be reduced by
conventional CD34+ selection, purging of PBSCs with simple CD34+
selection seems to be insufficient in CLL, due to the extensive
primary tumor load present in the majority of patients and the
possible existence of CD34+ CLL cells.
Both problems may be overcome, Dr. Dreger said, by adding a B-cell
depletion, or negative step, to the conventional CD34+ selection.
The German study involved 20 consecutive patients (mean age, 51
years) with poor-risk B-cell CLL who underwent stem cell mobilization
with chemotherapy and granulocyte colony-stimulating factor (G-CSF,
Double B-cell depletion of the harvested PBSC products was performed
using immunomagnetic CD34+ cell selection with the Isolex 300i
(Nexell Therapeutics, Inc., Irvine, California). This was followed by
a negative step with Nexells anti-CD 19/20/23/37-labeled
The purified PBSCs were reinfused after myeloablative treatment with
total body irradiation and high-dose cyclophosphamide. Patients were
followed by clinical and molecular methods.
We have added to the regular Isolex procedure consecutive tube
incubation of CD34+ products with Dynabeads that are prelabeled with
a cocktail of B-cell monoclonal antibodies against CD19/20/23/37
antigens from Nexell, Dr. Dreger said. The sensitized
residual B cells in the CD34+ products are then removed by magnetic
forces, and to further increase the purging efficacy, this negative
depletion step is repeated once, ending up with a
CD34+/B-cell-negative stem cell fraction.
A total of 25 separation runs was accomplished using collection
products containing 3.4% CD34+ cells and 1.2% CD19+/CD5+ CLL cells.
After double selection, 33% CD34+ cells were recovered with a purity
CLL cells were undetectable by high-resolution FACS (fluorescence
activated cell sorting) analysis in 15 of 25 final products, an
increase from 2 of 25 with positive purging only. The median purging
efficacy was a 5-log depletion (range, 4.1 to 6).
Evidence of CD34 enrichment could be seen in the increase of CD34+
cells from 3.4% in the starting fraction to 97.6% in the CD34+
selected fraction, and then to 98.8% in the final fraction, Dr.
The last six runs were performed with a refined separation software,
he said, resulting in an improvement of purity (98.8%) and recovery
(49% vs 37% with older software; P =.014) after the CD34+ selection
Overall, 1.2% of CLL cells were present in the starting fraction,
which was reduced to 0.05% after CD34+ selection, and then to 0%
after negative depletion, translating into a log depletion of 3.6
after CD34+ selection and finally to a log depletion of 5 after
All 20 patients proceeded to transplant with a median of 4.6 × 106
/kg CD34+ cells infused. Neutrophil recovery occurred at a mean of 9
days and platelet recovery at 10 days.
Rapid and durable engraftment occurred in all cases. At almost 1-year
median follow-up, there was one death due to pulmonary embolism 5
months post-transplant, one CLL progression 7 months post-transplant,
and one autoimmune thyroiditis 12 months post-transplant. One patient
experienced remission not only of CLL but also of psoriasis vulgaris,
which continued for more than 12 months.
We conclude that clinical scale positive/negative selection of
peripheral blood progenitor cells from patients with B-cell CLL with
the Isolex system results in about 5-log tumor cell depletion, and
that the reinfusion of these double-purged products into myeloablated
hosts is safe, Dr. Dreger said.