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 sarc oma. We used a new approach to purge artificially contaminated cellular spe cimens of Ewing's sarcoma and show the capacity for real-time polymerase ch ain reaction (PCR) to quantify the contamination level of Ewing's sarcoma i n 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 cy tometry. Using real-time PCR-based amplification of t(11;22), levels of Ewi ng's contamination of experimental and clinical cellular products were moni tored. Purging was accomplished using immunomagnetic-based depletion. Monit oring 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 hemato poietic 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 leve ls 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 sam ples is predicted to reduce tumor cell contamination to a level below the l imit of detection of PCR. Conclusion: These results demonstrate a new approach for purging contaminat ed cellular products of Ewing's sarcoma and demonstrate the capacity of rea ltime PCR to provide accurate quantitative estimates of circulating tumor b urden in this disease. J Clin Oncol 19:3649-3659. (C) 2001 by American Society of Clinical Oncolog y.