Follow‐up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome

Abstract

Bone marrow samples of 16 patients (two adults and 14 children) with a B lineage acute lymphoblastic leukaemia (ALL), and in whom Ig heavy chain gene rearrangements were detectable at diagnosis using polymerase chain reaction (PCR), were studied during evolution using PCR. The VDJ junctional fragment of the Ig heavy chain rearranged gene was amplified at diagnosis. After length reduction by restriction digestion, the amplified fragment was recovered by chromatography, labelled using a specific hexamer as a primer and directly used as a clonospecific probe. The sensitivity of the PCR ranged from 1:10(4) to 1:10(5) cells, depending on the patient's rearrangement. Residual disease (MRD) was detected in most of the patients achieving a complete remission after induction therapy, regardless of the long-term outcome of treatment. However, in patients remaining in complete remission, the level of MRD showed a tendency to decrease and ultimately become undetectable for variable periods of time, while in patients eventually relapsing there was a trend for MRD to persist at stable levels and even to increase before relapse was clinically evident. We conclude that the use of a simplified methodology for obtaining a clonospecific probe from the Ig heavy chain gene, though less sensitive than the sequencing methodology, is a valuable and readily available tool to monitor MRD in a high proportion of B lineage ALL.

Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging?

“…25,26 As long as the original rearranged band persisted, which in our hands corresponds to a percentage of at least 1%, no further analysis was performed and contamination was estimated using the value of the peak ( Figure 1). When no signal was observed (corresponding to fewer than 1% malignant cells), a semiquantitative PCR assay was done as described previously [21][22][23] to verify whether MCL cells were still detectable, the sensitivity of the method being 1:10 4 to 1:10 5 ( Figure 2). If residual malignant cells were identified, a LDA, derived from methods described by other authors 27,28 was performed, to better evaluate the number of residual lymphoma cells.…”

“…Detection of the IgH rearranged band at diagnosis: The DNA extracted from the original lymph node was amplified using different consensus VH primers: FR1c, 20 FR2c 20 or FR3c 21 and a JH consensus 3′ primer. 21 When amplification could be done with FR3/JH, a probe was directly made according to our previously described method.…”

“…21 When amplification could be done with FR3/JH, a probe was directly made according to our previously described method. [21][22][23] In parallel, sequencing of the CDR-III region was performed according to the method used in the laboratory 24 which consists of a direct fluorescence-based methodology with the Prism Sequenase Terminator Single-Stranded DNA Sequencing kit and the ABI 373 apparatus (Applied Biosystems, Foster City, CA, USA). This allowed designing an allele-specific oligomer (ASO) matching the D-N-J sequence for use in a limiting dilution analysis (LDA).…”

Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease

“…Sensitive and specific assays are needed to improve the detection of minimal residual disease (MRD) and direct the development of strategies for prevention of disease recurrence. Especially in ALL, the polymerase chain reaction (PCR) has been successfully applied to study remission bone marrow samples with high sensitivity (5,16,20,21,31,32). However, some approaches are not practical as performed on a routine basis, and in the vast majority of patients with AML, molecular markers that can be followed…”

Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR

“…With this approach, a detection limit of 5 × 10 −5 was reached in five cases and of 10 −3 in one case, which may not be sensitive enough for clinical application in ALL. 1,5,6,28,29 This problem has also been described in a recent paper by Pongers-Willemse et al, 13 who showed that this type of RQ PCR appeared to be comparable to quantification by dot-blotting but less sensitive than liquid hybridization. An additional disadvantage of this procedure is the expensive and time-consuming need for unique primer and probe sets for each individual patient.…”