Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA.
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.
Bartley
P A
PA
Haematology & Genetic Pathology, School of Medicine, Flinders University and Medical Centre, Bedford Park, SA, Australia.
Ross
D M
DM
Latham
S
S
Martin-Harris
M H
MH
Budgen
B
B
Wilczek
V
V
Branford
S
S
Hughes
T P
TP
Morley
Alexander A
AA
D000970:Antineoplastic Agents / Q000627:therapeutic use; D001549:Benzamides; D004273:DNA, Neoplasm / Q000032:analysis; D016044:Fusion Proteins, bcr-abl / Q000032:analysis* / Q000235:genetics; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000188:drug therapy; D018365:Neoplasm, Residual / Q000175:diagnosis*; D010879:Piperazines / Q000627:therapeutic use; D016133:Polymerase Chain Reaction / Q000379:methods; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D011743:Pyrimidines / Q000627:therapeutic use; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D001549:Benzamides; D004273:DNA, Neoplasm; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl