Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.

PURPOSE: 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. PATIENTS AND 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.
CONCLUSION: 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.