title: A DNA real-time quantitative PCR method suitable for routine monitoring of low levels of minimal residual disease in chronic myeloid leukemia.
abstract: 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 μg was assayed and 10(-7.0) when 80 μg 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.
authors.affiliation: Department of Haematology and Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Bartley
authors.affiliation: Department of Haematology and Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
authors.forename: Susan
authors.initials: S
authors.lastname: Latham
authors.affiliation: Department of Haematology and Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
authors.forename: Bradley
authors.initials: B
authors.lastname: Budgen
authors.affiliation: Department of Haematology and Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, South Australia, Australia; Haematology Division, SA Pathology, Adelaide, South Australia, Australia.
authors.forename: David M
authors.initials: DM
authors.lastname: Ross
authors.affiliation: Department of Haematology and Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hughes
authors.affiliation: Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia.
authors.forename: Susan
authors.initials: S
authors.lastname: Branford
authors.affiliation: South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia.
authors.forename: Deborah
authors.initials: D
authors.lastname: White
authors.affiliation: Haematology Division, SA Pathology, Adelaide, South Australia, Australia.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Hughes
authors.affiliation: Department of Haematology and Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, South Australia, Australia; Monoquant Pty. Ltd., Adelaide, South Australia, Australia. Electronic address: alec.morley@flinders.edu.au.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Morley
mesh_terms: D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*