Detection and Quantification of Minimal Residual Disease in Childhood B-Precursor Acute Lymphoblastic Leukemia by Limiting Dilution and PCR Application
Polymerase Chain Reaction (PCR) on Immunoglobine heavy chain and T-cellreceptor can be applied for the detection of Minimal Residual Disease (MRD) in children with B-precursor Acute Lymphoblastic Leukemia (ALL). We have previously shown that the presence of PCR detectable malignant cells at the end of induction therapy is strongly correlated with occurrence of a relapse. We currently investigate whether the predictive value of PCR can be increased by quantification of MRD during induction therapy.
A patient-specific nested PCR for tumorspecific Immunoglobine (IgH) and T-cell receptor gene (TCR) on two-fold diluted DNA samples (20 replicate experiments) is used in limiting dilution to detect leukemic cells. As this PCR can detect a single cell, the number of positive PCR-reactions at a certain dilution endpoint measures the number of residual leukemic cells using Poisson statistics. The applicability of this approach was evaluated on bone marrow samples taken at diagnosis and during remission of a child with oligoclonal (on Southern Blot analysis) B-precursor ALL.
At first remission the bone marrow contained residual ALL-cells between 4 × 10−3 and 5 × 10−4 cells. As multiple rearrangements were found in this oligoclonal patient, we tried to quantify each clone separately.
We were able to demonstrate the different behaviour in reduction during therapy of each clone. The clone which grew out during relapse, showed the slowest response during induction therapy.
We conclude that precise quantification of MRD during induction therapy can be investigated by application of a tumor-specific-IgH/TCRδ-PCR on serially diluted DNA samples. Based on the results of MRD detection for the various clones in this oligoclonal patient, we conclude it may be necessary to use multiple markers for MRD, also during induction therapy.
KeywordsPolymerase Chain Reaction Acute Lymphoblastic Leukemia Induction Therapy Minimal Residual Disease Clonal Rearrangement
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