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.
KeywordsStem Cell Tyrosine Hydroxylase Stem Cell Collection Stem Cell Product CD34 Selection
Unable to display preview. Download preview PDF.
- 1.Matthay KK, Villablanca JG, Seeger RC, Stram DO, Harris RE, Ramsay NK, Swift P, Shimada H, Black CT, Brodeur GM, Gerbing RB, Reynolds CP. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cisretinoic acid. N Engl J Med 1999;341:1165–1173.PubMedCrossRefGoogle Scholar
- 3.Freedman AS, Neuberg D, Mauch P, Soiffer RJ, Anderson KC, Fisher DC, Schlossman R, Alyea EP, Takvorian T, Jallow H, Kuhlman C, Ritz J, Nadler LM, Gribben JG. Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 1999;94:3325–3333.PubMedGoogle Scholar
- 5.Shpall EJ, Jones RB, Bearman SI, Franklin WA, Archer PG, Curiel T, Bitter M, Claman HN, Stemmer SM, Purdy M, Myers SE, Hami L, Taffs S, Heimfeld S, Hallagan J, Berenson RJ. Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment. J Clin Oncol 1994;12:28–36.PubMedGoogle Scholar
- 8.Brodeur GM, Pritchard J, Berthold F, Carlsen NLT, Castel V, Castleberry RP, De Bernardi B, Evans AE, Favrot M, Hedborg F, Kaneko M, Kemshead J, Lampert F, Lee REJ, Look AT, Pearson ADJ, Philip T, Roald B, Sawada T, Seeger RC. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993;11:1466–1477.PubMedGoogle Scholar
- 9.Grupp SA, Stern JW, Bunin N, Nancarrow C, Ross AA, Mogul M, Adams R, Grier HE, Gorlin JB, Shamberger R, Marcus K, Neuberg D, Weinstein HJ, Diller L. Tandem high dose therapy in rapid sequence for children with high-risk neuroblastoma. J Clin Onc 2000;18:2567–2575.Google Scholar
- 12.Donovan J, Temel J, Zuckerman A, Gribben J, Fang J, Pierson G, Ross A, Diller L, Grupp SA. CD34 selection as a stem cell purging strategy for neuroblastoma: pre-clinical and clinical studies. Med. Ped. Oncol. 2000;in press.Google Scholar
- 13.Lode HN, Handgretinger R, Schuermann U, Seitz G, Klingebiel T, Niethammer D, Beck J. Detection of neuroblastoma cells in CD34+ selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry. Eur J Cancer 1997;33:2024–2030.PubMedCrossRefGoogle Scholar
- 16.Greenfield D, Franklin WA, Tyson RW, Giller R, Shpall EJ. CD34 expression on pediatric solid tumors. Proc ASPHO 1996.Google Scholar
- 17.Tchirkov A, Kanold J, Giollant M, Halle-Haus P, Berger M, Rapatel C, Lutz P, Bergeron C, Plantaz D, Vannier JP, Stephan JL, Favrot M, Bordigoni P, Malet P, Briancon G, Demeocq F. Molecular monitoring of tumor cell contamination in leukapheresis products from stage IV neuroblastoma patients before and after positive CD34 selection. Med Pediatr Oncol 1998;30:228–232.PubMedCrossRefGoogle Scholar
- 18.Handgretinger R, Greil J, Schurmann U, Lang P, Gonzalez-Ramella O, Schmidt I, Fuhrer R, Niethammer D, Klingebiel T. Positive selection and transplantation of peripheral CD34+ progenitor cells: feasibility and purging efficacy in pediatric patients with neuroblastoma. J Hematofher 1997;6:235–242.CrossRefGoogle Scholar
- 19.Kanold J, Yakouben K, Tchirkov A, Halle P, Carret AS, Berger M, Rapatel C, deLumley L, Vannier JP, Plantaz D, LeGall E, Lutz P, Mechinaud F, Rialland X, Combaret V, Bordigoni P, Demeocq F. Long-term follow-up after CD34+ cell transplantation in children with neuroblastoma. Blood 1998;92:445a (abstr 1842).Google Scholar