LightCycler-based quantitative real-time PCR monitoring of patients with follicular lymphoma receiving rituximab in combination with conventional or high-dose cytotoxic chemotherapy.

OBJECTIVES: Quantitative real-time polymerase chain reaction (qPCR) is a suitable method to measure residual disease in hematological malignancies. Our objective was to assess a LightCycler-based qPCR for t(14;18)(q32;q21)(IgH/bcl-2)-positive cells quantification in the context of clinical and morphopathological characteristics of patients with follicular lymphoma treated with rituximab (R) in combination with conventional or high-dose chemotherapy. METHODS: A total of 270 bone marrow (BM) and peripheral blood (PB) samples collected from 52 patients with follicular lymphoma at diagnosis or at relapse before or sequentially during therapy were examined by qPCR and nested-PCR. RESULTS: A greater amount of t(14;18)-positive cells was observed in BM in comparison with PB in 76% of paired samples. The presence and number of t(14;18)-positive cells in BM and PB correlated with lymphoma activity. Significantly higher numbers of lymphoma cells were found in patients under non-remission compared with patients in clinical remission. During non-remission, 10-fold higher numbers were measured at relapse than at diagnosis. During remission, significantly higher levels were found in partial compared with complete remission. During first-line therapy, R/cyclophosphamide/adriamycin/vincristine/prednisone (CHOP) had higher in vivo purging ability than R/fludarabine/mitoxantrone (FM). After R/high-dose cytosine-arabinoside and mitoxantrone (HAM) or R/carmustine/etoposide/cytarabine/melphalan (BEAM), the level of t(14;18)-positive cells dropped below the detection limit in 80% of patients. CONCLUSIONS: LightCycler qPCR is a reliable method for quantitative molecular monitoring of t(14;18)-positive cells in BM and PB of patients with follicular lymphoma. It reflects the clinical characteristics of patients and allows assessment of response to different treatment regimens on a molecular level.

Levels of minimal residual disease detected by quantitative molecular monitoring herald relapse in patients with multiple myeloma.

BACKGROUND AND OBJECTIVES: Detection of minimal residual disease (MRD) has helped to improve the treatment of patients with leukemia. At present MRD testing in patients with multiple myeloma (MM) is not applied as a standard diagnostic or prognostic method. DESIGN AND METHODS: Immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) using patient-specific TaqMan probes together with LightCycler technology was performed to quantify minimal residual disease in MM. Relative levels of clonotypic cells were assessed as IgH/2beta-actin ratios with a sensitivity of 10(-4) to 10(-5). RESULTS: Following stem cell transplantation, a significant reduction of clonotypic cells was observed in bone marrow (BM) and peripheral blood (PB) samples of 11 patients, comparing pre-treatment values with those of best response (median: 13% to 0.09% and 0.03% to 0%, respectively). In 5 patients with ongoing clinical remission IgH/2beta-actin ratios remained stable at a low level, while in 6 patients an increase to 2% in BM and 0.4% in PB was associated with progression of the disease. In 4 of these 6 patients the increase of clonotypic cells in PB was detectable a median of 3 months (range: 0.5-6) before relapse. Furthermore, time-to-progression of patients with pre-transplantation IgH/2b-actin ratios > 0.03% in BM was significantly shorter than that of patients with lower MRD levels. INTERPRETATION AND CONCLUSIONS: MRD in patients with MM can be quantified reliably using TaqMan chemistry adapted to the LightCycler system. Residual tumor cell levels before transplantation as well as results of sequential molecular monitoring are predictive of relapse.