Around 50% of chronic myeloid leukaemia (CML) patients who remain on imatinib treatment for more than 5 years will achieve a complete molecular response (CMR), defined by undetectable BCR-ABL mRNA in a sensitive reverse transcriptase real-time quantitative PCR (RQ-PCR) assay. Given the increasing importance of CMR on imatinib therapy the primary aim of this study was to improve the accuracy and sensitivity of MRD detection to allow a more accurate estimation of relapse risk when therapy is withdrawn. Firstly, we investigated ways of improving the sensitivity of RT-PCR methods for the detection of BCR-ABL mRNA. Secondly, we investigated the use of the patient-specific BCR-ABL gene for the detection of MRD. Thirdly, we conducted a multi-centre clinical trial of imatinib withdrawal in selected CML patients in a stable CMR. This clinical trial provided patient samples that could be used to test our optimized MRD assays, and provided clinical data on the risk and patterns of relapse after withdrawal of imatinib therapy. The trial is ongoing, but an interim analysis of the study data was performed. In 22 patients the estimated probability of molecular relapse after imatinib withdrawal was 54%, and 60% of relapses occurred within the first 4 months. The average detection limit of BCR-ABL mRNA by RQ-PCR is estimated at around 4.5 log below the level of BCR-ABL prior to commencing treatment. The number of leukaemic cells at diagnosis is around 10¹ ², so the number of residual leukaemic cells in CMR might vary from zero to over a million. We hypothesized that the amount of residual leukaemia in CMR is variable between patients, and that this heterogeneity is a determinant of the risk of relapse when treatment is withdrawn. We developed more sensitive methods for the detection of BCR-ABL and tested these methods in samples from our study patients. We showed that random pentadecamer (15-mer) primers improved the efficiency of reverse transcriptase PCR (RT-PCR), and resulted in a lower detection limit of BCR-ABL mRNA. We also developed a novel nested RT-PCR method using real-time PCR for the second round of the reaction, and this resulted in a lower detection limit of BCR-ABL in patient samples. The utility of this nested RT-PCR method was limited by a false positive rate of 2-3% in the HeLa cell line that we used as our negative control. Consequently, we examined the detection of the patient-specific genomic BCR-ABL sequence as an alternative to RT-PCR. Breakpoints in BCR and ABL1 in CML patients are widely dispersed over 3 kb and 150 kb, respectively. Therefore, the BCR-ABL genomic sequence is essentially unique to each patient. We sequenced the genomic breakpoints of 43 CML patients. We showed that the distribution of breakpoints in BCR and ABL1 was non-random, but we were unable to identify any genomic feature that determined the specific location of individual breakpoints. We developed a novel BCR-ABL DNA Q-PCR method for 12 of the study patients, and in 11 of the patients BCR-ABL DNA was detected when the patient was in a CMR, confirming that this method was more sensitive than RQ-PCR. Contrary to our hypothesis, the detection of BCR-ABL DNA was not predictive of relapse. In most patients who relapsed there was a significant increase in BCR-ABL DNA prior to mRNA relapse. Two patients had stable levels of BCR-ABL DNA measurable on multiple occasions, but remained in remission after 6 months and 15 months, respectively. We have shown that a stable CMR after the withdrawal of imatinib therapy does not necessarily indicate the eradication of leukaemia. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010
| Identifer | oai:union.ndltd.org:ADTP/288073 |
| Date | January 2010 |
| Creators | Ross, David Morrall |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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