A real-time PCR assay for the quantification of residual malignant cells in B cell chronic lymphatic leukemia

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Abstract
Several new therapeutic approaches for the treatment of monoclonal B cell lymphomas are currently being investigated. In parallel with new therapeutic modalities, more sensitive diagnostic methods are needed. These methods should be highly sensitive in detecting very low amounts of malignant cells and should be specific for the malignant clone. In addition, these methods should allow the quantification of residual tumor cells. In this study a new real-time polymerase chain reaction (LightCycler) was evaluated to quantify residual tumor cells in monoclonal B cell malignancies. This technology combines the advantages of rapid cycling PCR with the online detection of PCR products using fluorescent dyes. Our assay is based on immunoglobulin heavy chain (IgVH)-specific PCR with allele-specific primers complementary to hypervariable CDRII and CDRIII regions. A set of framework region III (FRIII)-specific hybridization probes was used for detection of the specific amplification product, and IgVH copy number was quantified with the cloned IgVH sequence as an external standard. The approach was evaluated with the Hodgkin lymphoma cell line L428 in order to quantify L428 dilutions. L428 cells mixed with peripheral blood mononuclear cells (PBMNCs) were detected and quantified with a sensitivity of one cell within 1 × 105 PBMNCs. Sample DNA from the peripheral blood and from the bone marrow of two patients with B-CLL was analyzed in the new set up at different time points before and after therapy. Statistically significant changes in IgVH copy numbers were documented in both patients. We conclude that this technology offers an additional opportunity to detect and quantify residual tumor cells in B-CLL over several log steps with a high sensitivity. The kinetics of residual tumor cell counts in B-CLL can be analyzed by this method.