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Quantitation of leukemia clone-specific antigen gene rearrangements by a single-step PCR and fluorescence-based detection method
- Source :
- Leukemia. 13:1843-1852
- Publication Year :
- 1999
- Publisher :
- Springer Science and Business Media LLC, 1999.
-
Abstract
- PCR-based detection of minimal residual disease (MRD) in children with precursor B cell acute lymphoblastic leukemia (pre-B ALL) by amplification of clone-specific antigen gene rearrangements has been labor-intensive. In this study we present a simpler, yet accurate, method to assess and quantitate MRD in pediatric pre-B ALL that utilizes these markers. From the sequence of the immunoglobulin heavy chain (IgH) and T cell receptor (TcR) gene rearrangements characterized at the time of diagnosis or relapse, primers were designed and tested in a clone-specific, single-round PCR assay, then analyzed by fluorescence staining of the PCR products. The most critical step involved an adherence to a new set of guidelines for the design of clone-specific primers. Application of this method to 54 IgH and 13 TcR (nine Vdelta2Ddelta3 and four Ddelta2-Ddelta3) gene rearrangements in 47 patients resulted in an intense band within the region of the predicted molecular weight, confirming the reproducibility of the assay. Quantitative applications of the approach were examined by performing a 10-replicate limiting dilution clone-specific PCR on six diagnostic samples and an asymptotic response model of the Von Krogh form was found to fit the data well. From this model, estimation of leukemic cells of remission bone marrow samples was achieved at a detection sensitivity of 2 x 10(-6). The method is demonstrated on 18 patients whose marrows were prospectively analyzed during therapy. We conclude this methodology is useful in the quick and accurate assessment of MRD in children with pre-B ALL, and could be applied to other DNA quantitation assays.
- Subjects :
- Cancer Research
Neoplasm, Residual
DNA Mutational Analysis
Receptors, Antigen, T-Cell
Bone Marrow Cells
Biology
Polymerase Chain Reaction
Sensitivity and Specificity
Fluorescence
law.invention
Antigens, Neoplasm
law
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma
Acute lymphocytic leukemia
Biomarkers, Tumor
medicine
Humans
Prospective Studies
Child
Antigen Gene
Polymerase chain reaction
DNA Primers
Gene Rearrangement
Clinical Trials as Topic
Base Sequence
Genes, Immunoglobulin
Remission Induction
T-cell receptor
Hematology
Gene rearrangement
medicine.disease
Burkitt Lymphoma
Molecular biology
Minimal residual disease
Clone Cells
Leukemia
Oncology
Immunology
Immunoglobulin heavy chain
Subjects
Details
- ISSN :
- 14765551 and 08876924
- Volume :
- 13
- Database :
- OpenAIRE
- Journal :
- Leukemia
- Accession number :
- edsair.doi.dedup.....6eb5bcc5392275c28bed753add0359df
- Full Text :
- https://doi.org/10.1038/sj.leu.2401530