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Immunomagnetic Purging of Ewing’s Sarcoma From Blood and Bone Marrow: Quantitation by Real-Time Polymerase Chain Reaction

From the Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, and Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD; Department of Pediatrics, Walter Reed Army Medical Center, Washington, DC; and Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY.

Address reprint requests to Margret E. Merino, MD, Pediatric Oncology Branch, National Cancer Institute, National Institute of Health, 9000 Rockville Pk, Bethesda, MD 20892-1928; email: merinom{at}mail.nih.gov

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 10⁶ 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 1:10⁶. Levels of contamination in clinical samples ranged from 1:10⁵ to 10⁶. 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.

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