Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.
OBJECTIVE
A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens.


METHODS
Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays.


RESULTS
MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR.


CONCLUSIONS
These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.
Merino
M E
ME
Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1928, USA. merinom@mail.nih.gov
Navid
F
F
Christensen
B L
BL
Toretsky
J A
JA
Helman
L J
LJ
Cheung
N K
NK
Mackall
C L
CL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D001853:Bone Marrow / Q000473:pathology; D016465:Bone Marrow Purging / Q000592:standards; D017931:DNA Primers; D005434:Flow Cytometry; D006801:Humans; D018189:Immunomagnetic Separation / Q000592:standards*; D007937:Leukapheresis / Q000592:standards; D007963:Leukocytes, Mononuclear / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000473:pathology* / Q000628:therapy*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017931:DNA Primers