Successful Purging of Stem Cell Products Using CD34 Selection
Flow cytometric detection of CD34 on NB cell lines. We assessed CD34 expression using a panel of anti-CD34 monoclonal antibodies including 9C5, 12.8 and QBend10 and showed no increase in labeling over secondary-only control.
Spiking experiments with the Isolex 50 system. NB cell lines were used to contaminate aliquots of stem cell collections, after which the products were purified using the Isolex 50. Purging of NB was assessed by quantitative multiplex RT-PCR (Taqman system) using a tumor-specific transcript, GAGE. We demonstrated <2 logs of tumor cell depletion from these specimens.
Analysis of clinical specimens. Stem cell pre- and post-CD34 selection were analyzed from patients treated on a tandem transplant trial for NB. In nine specimens selected using the Ceprate LC CD34 selection system where tumor was detectable by immunocytochemistry pre-selection, we observed >2.4 to >4.6 logs of NB purging after selection. We then analyzed 23 aliquots of stem This research was supported in part by the University of Pennsylvania Cancer Center (SG), by the Benacerraf/Frei Clinical Investigator Award, Dana-Farber Cancer Institute (LD) and the Fiftieth Anniversary Program for Scholars in Medicine, Harvard Medical School (LD). The abbreviations used are: FITC, fluorescein isothiocyanate; GAPDH, Glyceraldehyde 3 phosphate dehydrogenase; ICC, immunocytochemistry; MoAbs, monoclonal antibodies; PE, phycoerythrin; RT-PCR, reverse transcriptase-polymerase chain reaction; PBMC, peripheral blood mononuclear cells.
cells infused into patients post-CD34 selection and compared to the product pre-selection. 20/23 specimens showed depletion of NB, although some level of GAGE message was observed in most post-CD34 selection specimens. These data show that purging of NB from stem cells using CD34 selection is feasible, yielding infused products that are negative at the level of ICC but often positive at the level of RT-PCR.
KeywordsToxicity Lymphoma Platinum Tyrosine Leukemia
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