A validated real-time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients

Objective - Purging procedures are increasingly used to provide stem cell collections devoid of contaminating tumor cells. In follicle center lymphoma (FCL), most approaches eradicate polymerase chain reaction (PCR);-detectable disease in only a fraction of harvests undergoing ex vivo manipulation. In this study we evaluated whether there is a relationship between tumor burden of stem cell harvests and successful clearance of PCR-detectable disease following ex vivo manipulation. Materials and Methods - To address this issue, we developed a real-time PCR approach for quantitative measurement of tumor contamination using the bcl-2 rearrangement. Real-time PCR was used to evaluate the relationship between tumor burden of stem-cell harvests and purging effectiveness in PCR⁺ samples derived from 10 FCL patients. Ex vivo purging was performed using the MaxSep cell separator (Baxter Immunotherapy, Deerfield, IL, USA). Results - Our real-time PCR method proved effective, sensitive, accurate, and reproducible. Four collections were successfully cleared of minimal residual disease (MRD) whereas six remained PCR⁺. Real-time PCR showed that the four collections successfully cleared of MRD had a prepurging tumor burden significantly lower than those remaining PCR⁺ (p = 0.04). Conclusion - This study provides the first evidence that evaluation of tumor burden in stem-cell harvests by real-time PCR can predict the effectiveness of therapeutic intervention in non-Hodgkin's lymphoma. Based on these findings, we foresee a more widespread use of this technique to evaluate the impact of different therapeutic approaches in FCL.