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

Several new therapeutic approaches for the treatment of monoclonal B cell l ymphomas are currently being investigated. In parallel with new therapeutic modalities, more sensitive diagnostic methods are needed. These methods sh ould be highly sensitive in detecting very low amounts of malignant cells a nd should be specific for the malignant clone. In addition, these methods s hould allow the quantification of residual tumor cells. In this study a new real-time polymerase chain reaction (LightCycler) was evaluated to quantif y residual tumor cells in monoclonal B cell malignancies. This technology c ombines the advantages of rapid cycling PCR with the online detection of PC R products using fluorescent dyes. Our assay is based on immunoglobulin hea vy chain (IgVH)-specific PCR with allele-specific primers complementary to hypervariable CDRII and CDRIII regions. A set of framework region III (FRII I)-specific hybridization probes was used for detection of the specific amp lification product, and IgVH copy number was quantified with the cloned IgV H sequence as an external standard. The approach was evaluated with the Hod gkin lymphoma cell line L428 in order to quantify L428 dilutions. L428 cell s mixed with peripheral blood mononuclear cells (PBMNCs) were detected and quantified with a sensitivity of one cell within 1x10(5) 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 afte r therapy. Statistically significant changes in IgVH copy numbers were docu mented in both patients. We conclude that this technology offers an additio nal opportunity to detect and quantify residual tumor cells in B-CLL over s everal log steps with a high sensitivity. The kinetics of residual tumor ce ll counts in B-CLL can be analyzed by this method.