A DNA Real-Time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia

The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid Leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures. PCR primers were chosen from a library of presynthesized and pretested BCR (n = 19) and ABL1 (n = 568) primers. BCR-ABL1 sequences were quantified relative to BCR sequences in 521 assays on 266 samples from 92 patients. For minimal residual disease detectable by DNA qPCR and RT-qPCR, DNA qPCR gave similar minimal residual disease results as RT-qPCR but had better precision at low minimal residual disease Levels. The Limit of detection of DNA qPCR depended on the amount of DNA assayed, being 10(-5.8) when 5 mu g was assayed and 10(-7.0) when 80 mu g was assayed. DNA qPCR may be useful and practical for monitoring the increasing number of patients with minimal residual disease around or below the limit of detection of RT-qPCR as the assay itself is simple and the up-front costs will be amortized if sequential assays are performed.