Quantitative molecular monitoring of residual tumor cells in chronic lymphocytic leukemia

Abstract.

New therapeutic approaches for the treatment of B-cell chronic lymphocytic leukemia (B-CLL) can induce remarkable responses. Molecular remissions have been observed occasionally after high-dose chemotherapy. Thus, new improved techniques to monitor residual tumor cells on a molecular basis in CLL are warranted. For this purpose, a real-time quantitative allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) for patients with B-CLL was designed. In the present study, the PCR assay was standardized with identical cycling parameters as well as primer, probe, and MgCl₂ concentration for each patient. Ten patients were monitored with DNA samples obtained at 52 time points (median: 5.2 per patient). The median follow-up per patient was 11.4 months. Nine of ten patients had PCR-detectable residual tumor cells in the peripheral blood after therapy. One patient became PCR negative with a combination of fludarabine and rituximab after the end of treatment. The MRD levels in patients with detectable disease ranged from 0.002% to 10.1% after therapy. We conclude that real-time quantitative ASO-PCR can be utilized for quantitative molecular monitoring of minimal residual disease (MRD) in B-CLL patients in complete remission (CR), that new effective treatment approaches such as combined chemo/immunotherapy can render CLL patients PCR negative, and that different MRD levels in PCR-positive patients were observed warranting further investigation into possible correlation with clinical outcome.