Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA

Paul Bartley, David Ross, Susan Latham, Michael Martin-Harris, Bradley Budgen, Vicki Wilczek, S Branford, Timothy Hughes, Alexander Morley

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    43 Citations (Scopus)

    Abstract

    Increasing numbers of patients with chronic myeloid leukaemia (CML) treated with tyrosine kinase inhibitors achieve undetectable levels of BCR-ABL mRNA using sensitive quantitative real-time reverse transcriptase PCR (RT-qPCR) methods and a method to measure minimal residual disease (MRD) in patients with low levels could be of value. Following isolation and sequencing of the patient-specific BCR-ABL breakpoint, a DNA-based nested qPCR assay was established, and MRD was measured by this method and one-round RT-qPCR in 38 samples from 24 patients with CML. Mixing experiments using patient DNA in normal DNA indicated that DNA qPCR could detect BCR-ABL sequences at a limit of approximately 10-6. In 22 samples in which MRD was detectable by both methods, comparison of the results of DNA qPCR with the results obtained on the same sample by RT-qPCR showed good correlation. In another 16 samples, BCR-ABL mRNA was not detectable by RT-qPCR. In 8 of the 16 samples, BCR-ABL DNA was detected at levels ranging from 1.1 × 10-5 up to 2.8 × 10-4 and in the remaining eight samples BCR-ABL was not detected by either method. In one patient, who had stopped imatinib, an almost 1000-fold rise in MRD, to 5.2 × 10-4 was observed in sequential samples. Nested DNA qPCR was more sensitive than one-round RT-qPCR and could be used for the monitoring of patients with CML with very low levels of MRD.

    Original languageEnglish
    Pages (from-to)e222-e228
    Number of pages7
    JournalInternational Journal of Laboratory Hematology
    Volume32
    Issue number6 PART 1
    DOIs
    Publication statusPublished - Dec 2010

    Keywords

    • BCR-ABL
    • chronic myeloid leukaemia
    • Minimal residual disease
    • monitoring
    • PCR

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