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

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 allelespecific 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 × 10 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 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. Leukemia (2000) 14, 754-766.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation

“…Thus, our data demonstrate higher sensitivity of ASO IgH RQ-PCR compared to our MRD flow assay in post-transplant CLL patients. This contradicts in vitro data that had predicted sensitivities of ASO IgH RQ-PCR [21][22][23] within the same order of magnitude as for MRD flow approaches (one CLL cell in 10 4 -10 5 normal cells). 18,25,29,30 We therefore analyzed factors that influence specificity and sensitivity of MRD flow as the apparently less sensitive method.…”

“…It has been reported that MRD determinations by RQ-PCR at a level of 1E-4 resulted in a coefficient of variation (CV) of 32%. 21 For MRD flow (like for all rare cell measurements by flow cytometry 48 ), the CV is dependent on the absolute number of CLL cells acquired. Although we did not formally determine the retest reliability of our assay, at the absolute specificity threshold (20 CLL cells), a CV of about 23% is expected.…”

“…14,17,19,20 Real-time PCR using such ASO primers allows the quantitation of one CLL cell in 10 4 -10 5 cells. [21][22][23] A more rapid, but less sensitive and not quantitative method for MRD detection in CLL is PCR using consensus IgH-primers (consensus IgH-PCR). 12,16,18,24,25 This technique is reported to detect one monoclonal B-cell in 2 Â 10 1 -1 Â 10 3 benign leukocytes.…”

Stem cell transplantation for chronic lymphocytic leukemia: different outcome after autologous and allogeneic transplantation and correlation with minimal residual disease status

“…Moreover, as recently shown, real-time PCR assays may be helpful in analyzing the MRD kinetics. 47,48 In summary, the present report suggests that in CLL patients the probability of obtaining sustained MRD(−) CR after autotransplantation is low and that MRD analysis is useful to predict clinical relapse. On the other hand, the long-lasting responses observed after allogeneic transplants indicate the possibility of a true eradication of CLL in a significant proportion of patients, although delayed responses or transient relapses at MRD level may be detected in some instances.…”

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

“…17,20 In the last 4 years, RQ-PCR has become an attractive and useful approach for the detection of MRD in immature B-cell malignancies and the subset of B-CLL without somatic mutations, 17 mainly because of the utilization of consensus probes in the JH region thereby avoiding the need to synthesize allele-specific probes (ASO probes) for each patient, which is very expensive and time-consuming. 18,31 Unfortunately, this approach is less optimal for MRD analysis of postgerminal center derived-B-cell malignancies containing a high level of SH, because of mismatches between the probes/ primers and the mutated VDJH rearrangement. Ladetto et al 14 has suggested that zero or one point mutation in the probe binding site does not influence the effectiveness of the RQ-PCR, and the presence of three or more point mutations in that site is always associated with failure of the RQ-PCR.…”