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David M. Ross, Ilaria S. Pagani, Yazad D. Irani, Jade Clarson, Tamara Leclercq, Phuong Dang, Jennifer McLean, Verity A. Saunders, Lisa Carne, John Reynolds, David S. Ritchie, Deborah L. White, Susan Branford, Timothy P. Hughes, Agnes S. M. Yong

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.

We have developed a rapid real-time quantitative PCR method for measuring BCR-ABL mRNA levels in peripheral blood in chronic myeloid leukaemia (CML). The technique was used to monitor minimal residual disease for the early detection of relapse and as an assessment of treatment response. Normal BCR mRNA was quantitated to control for RNA degradation and the results reported as a percentage of BCR-ABL/BCR. Every patient measured at diagnosis (n = 21) had increased expression of BCR-ABL of up to 5-fold above the normal BCR levels. With effective treatment the BCR-ABL levels decreased. The molecular data was correlated with Philadelphia chromosome levels in bone marrow and a good correlation was found when treatment induced a cytogenetic response (Spearman correlation = 0.94, P < 0.0001, n = 67 samples). In patients receiving interferon-alpha therapy we found a significant difference in the BCR-ABL levels between cytogenetic response groups. The method was sensitive, reproducible, and readily detected a change in BCR-ABL transcript levels in serial blood samples. Sample throughput was high because post PCR processing was unnecessary. We conclude that real-time quantitative PCR monitoring of peripheral blood can be used to reliably monitor disease response in CML.

An atypical BCR-ABL transcript in a patient with Philadelphia (Ph)-positive chronic myeloid leukaemia (CML) was detected using a long polymerase chain reaction technique. Sequence analysis revealed a joining of BCR exon e8 and ABL exon a2, with a 55-base pair (bp) insert of an inverted sequence of ABL intron 1b between them, giving rise to an in frame e8a2 BCR-ABL transcript. This is the first report of a BCR-ABL transcript with the insertion of an inverted sequence. The long PCR technique is a useful screen for atypical BCR-ABL transcripts not detected by standard techniques.