Paul A. Bartley

Patients with chronic myeloid leukemia who maintain a complete molecular response after stopping imatinib treatment have evidence of persistent leukemia by DNA PCR

Around 40–50% of patients with chronic myeloid leukemia (CML) who achieve a stable complete molecular response (CMR; undetectable breakpoint cluster region-Abelson leukemia gene human homolog 1 (BCR–ABL1) mRNA) on imatinib can stop therapy and remain in CMR, at least for several years. This raises the possibility that imatinib therapy may not need to be continued indefinitely in some CML patients. Two possible explanations for this observation are (1) CML has been eradicated or (2) residual leukemic cells fail to proliferate despite the absence of ongoing kinase inhibition. We used a highly sensitive patient-specific nested quantitative PCR to look for evidence of genomic BCR–ABL1 DNA in patients who sustained CMR after stopping imatinib therapy. Seven of eight patients who sustained CMR off therapy had BCR–ABL1 DNA detected at least once after stopping imatinib, but none has relapsed (follow-up 12–41 months). BCR–ABL1 DNA levels increased in all of the 10 patients who lost CMR soon after imatinib cessation, whereas serial testing of patients in sustained CMR showed a stable level of BCR–ABL1 DNA. This more sensitive assay for BCR–ABL1 provides evidence that even patients who maintain a CMR after stopping imatinib may harbor residual leukemia. A search for intrinsic or extrinsic (for example, immunological) causes for this drug-free leukemic suppression is now indicated.

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.

Incorporation of measurement of DNA integrity into qPCR assays

Optimal accuracy of quantitative PCR (qPCR) requires correction for integrity of the target sequence. Here we combine the mathematics of the Poisson distribution and exponential amplification to show that the frequency of lesions per base (which prevent PCR amplification) can be derived from the slope of the regression line between cycle threshold (Ct) and amplicon length. We found that the amplifiable fraction (AF) of a target can be determined from this frequency and the target length. Experimental results from this method correlated with both the magnitude of a damaging agent and with other measures of DNA damage. Applying the method to a reference sequence, we determined the values for lesions/base in control samples, as well as in the AFs of the target sequence in qPCR samples collected from leukemic patients. The AFs used to calculate the final qPCR result were generally >0.5, but were <0.2 in a few samples, indicating significant degradation. We conclude that DNA damage is not always predictable; quantifying the DNA integrity of a sample and determining the AF of a specific qPCR target will improve the accuracy of qPCR and aid in the interpretation of negative results.

Durable complete molecular remission of chronic myeloid leukemia following dasatinib cessation, despite adverse disease features

Patients with chronic myeloid leukemia, treated with imatinib, who have a durable complete molecular response, might remain in complete molecular response after stopping treatment. Previous reports of patients stopping treatment in complete molecular response have included only patients with a good response to imatinib. We describe 3 patients with stable complete molecular response on dasatinib treatment following imatinib failure. Two of the 3 patients remain in complete molecular response more than 12 months after stopping dasatinib. In these 2 patients we used highly sensitive patient-specific BCR-ABL1 DNA PCR to show that the leukemic clone remains detectable, as we have previously shown in imatinib-treated patients. Dasatinib-associated immunological phenomena, such as the emergence of clonal T-cell populations, were observed both in one patient who relapsed and in one patient in remission. Our results suggest that the characteristics of complete molecular response on dasatinib treatment may be similar to that achieved with imatinib, at least in patients with adverse disease features.

Rapid isolation of translocation breakpoints in chronic myeloid and acute promyelocytic leukaemia

Isolation and sequencing of the translocation breakpoint in chronic myeloid leukaemia‐(CML) and acute promyelocytic leukaemia (APML) may help to elucidate the mechanism of translocation and provide a molecular marker for monitoring of minimal residual disease. Amplification across the translocation breakpoint was performed in samples from 91 patients with CML and 15 patients with APML using single‐tube multiplex polymerase chain reaction (PCR) involving 308 primers for CML and 40 primers for APML. Nonspecific amplification was removed by a modification of PCR, termed sequential hybrid primer PCR (SHP‐PCR), which involved two sequential rounds of PCR, each of which used a low concentration of a specially designed hybrid primer. The resultant amplified material was sequenced. The method as finally developed was simple quick and robust. The translocation breakpoint was successfully isolated and sequenced in all 106 samples. The strategy of highly multiplexed PCR followed by SHP‐PCR is thus an effective method for isolating the translocation breakpoint in CML and APML. It may also be applicable to other haematological disorders characterised by translocation, deletion or inversion.

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 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.

Distribution of genomic breakpoints in chronic myeloid leukemia: Analysis of 308 patients

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BCR-ABL1 expression, RT-qPCR and treatment decisions in chronic myeloid leukaemia

Aims RT-qPCR is used to quantify minimal residual disease (MRD) in chronic myeloid leukaemia (CML) in order to make decisions on treatment, but its results depend on the level of BCR-ABL1 expression as well as leukaemic cell number. The aims of the study were to quantify inter-individual differences in expression level, to determine the relationship between expression level and response to treatment, and to investigate the effect of expression level on interpretation of the RT-qPCR result. Methods BCR-ABL1 expression was studied in 248 samples from 65 patients with CML by determining the difference between MRD quantified by RT-qPCR and DNA-qPCR. The results were analysed statistically and by simple indicative modelling. Results Inter-individual levels of expression approximated a normal distribution with an SD of 0.36 log. Expression at diagnosis correlated with expression during treatment. Response to treatment, as measured by the number of leukaemic cells after 3, 6 or 12 months of treatment, was not related to the level of expression. Indicative modelling suggested that interpretation of RT-qPCR results in relation to treatment guidelines could be affected by variation in expression when MRD was around 10% at 3 months and by both expression variation and Poisson variation when MRD was around or below the limit of detection of RT-qPCR. Conclusions Variation between individuals in expression of BCR-ABL1 can materially affect interpretation of the RT-qPCR when this test is used to make decisions on treatment.

Sensitive monitoring of acute promyelocytic leukemia by PML-RARA DNA Q-PCR

Ivar O. Kommers, Paul A. Bartley, Bradley Budgen, Sue Latham, Ashanka Beligaswatte, Shane G. Supple, Alberto Catalano, Harry J. Iland, Alexander A. Morley and David M. Ross Department of Clinical and Molecular Medicine, Flinders University and Medical Centre, Adelaide, Australia; VU University Medical Center, Amsterdam, The Netherlands; Institute of Haematology, Royal Prince Alfred Hospital, Sydney, Australia; Sydney Medical School, University of Sydney, Sydney, Australia; School of Medicine, University of Adelaide, Adelaide, Australia