REVIEWMolecular monitoring of BCR–ABL as a guide to clinical management in chronic myeloid leukaemia
Introduction
In 2005 most chronic myeloid leukaemia (CML) patients in chronic phase will receive imatinib mesylate as their first line of therapy. This is a remarkable development considering it was only 7 years ago that the first CML patient received imatinib1, 2, 3, 4. The rapid change in medical practice has been driven by the excellent response rates and good tolerability observed in chronic phase CML. A key trial in establishing the superiority of imatinib for chronic phase CML patients was the IRIS trial, a randomised comparison of imatinib versus interferon alpha plus low dose Ara-C5. This demonstrated that the vast majority of patients treated with imatinib achieve a stable complete cytogenetic response (CCR). Patients achieving this level of response have a low risk of progression over the next 24 months. These and other results showing generally excellent and stable responses6, 7, 8, 9, 10, 11, 12, 13 has led some clinicians to become less vigilant than they used to be regarding the need for regular cytogenetic and/or molecular assessments of response. However, there are significant concerns regarding imatinib monotherapy in the management of CML that justify a policy of frequent accurate molecular monitoring. Primary refractoriness and acquired resistance are each seen in 5–10% of de novo CML patients on imatinib5. These patients should be identified early in their disease course when intervention may still be effective. In addition, the majority of good responders have evidence of persistent leukaemia by real-time quantitative PCR (RQ-PCR) even after 2–3 years of therapy14, 15 suggesting the potential for resistance in the long term and the need for ongoing molecular monitoring even in the best responders.
Other ABL kinase inhibitors have recently been developed and are in early clinical trials16. These agents may be effective in some cases of primary or secondary resistance. Second generation ABL kinase inhibitors offer potential advantages in terms of greater potency against BCR–ABL relative to other targets and improved activity against some imatinib-resistant mutants. Defining the role of these inhibitors in specific cases of imatinib-resistance will be highly dependent on early recognition of emerging imatinib-resistance and, if possible, full characterisation of the cause.
The critical causative event in CML is the formation of the Philadelphia chromosome (Ph) which results in the fusion of the head of the BCR gene with the body of the ABL gene17. This results in the production of a fusion transcript and protein18. The c-Abl protein is predominantly present in the nucleus and plays a key role in cell cycle control. It is a tightly regulated tyrosine kinase. The fused oncoprotein BCR–ABL is mainly present in the cytoplasm and is an activated tyrosine kinase19. Signal transduction pathways that are vital for the regulation of normal haematopoiesis are constitutively activated in BCR–ABL expressing cells, which leads to deregulated proliferation, differentiation and survival. Retrovirally expressed BCR–ABL in murine marrow cells can induce a CML like disorder confirming that it is necessary and sufficient to cause CML20, 21.
Imatinib is a specific tyrosine kinase inhibitor that inhibits the kinase activity of Bcr-Abl, Abl, ABL-related gene (Arg), platelet derived growth factor receptor (PDGF-R) and the Kit receptor22, 23, 24. Imatinib was originally thought to act as a direct competitive inhibitor of ATP binding. However, structural resolution of imatinib with a compound closely resembling imatinib showed that it occupies only part of the ATP binding pocket25, 26. Imatinib exploits the distinctive inactive conformation of the Bcr-Abl activation loop and consequently achieves high specificity.It contacts 21 amino acids within the ATP binding site and the activation loop. The protein is tightly held in the inactive state which prevents ATP binding and the phosphorylation of Bcr-Abl. Consequently the downstream effector molecules are not phosphorylated and the signal transduction pathways are not activated.
Numerous somatic point mutations within the BCR–ABL kinase domain have been described that impair imatinib binding while not affecting ATP binding27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43. These mutations are the commonest cause of acquired imatinib resistance30, 31, 32, 37, 38, 44, 45, 46.
Cytogenetic analysis has been the mainstay of disease monitoring in CML. Response criteria based on the percentage of Ph-positive cells in the bone marrow were established for patients on interferon alpha. They have proved to be good predictors of long term response47, 48. Over the past 12 years, several groups have developed quantitative RT-PCR assays to measure BCR–ABL transcript levels in the blood and marrow that enabled the dynamics of residual disease to be monitored over time and has provided a viable alternative for disease monitoring49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69. The transcript level correlates with the number of leukaemic cells present in the blood and marrow and can be used as an accurate barometer of the response to therapy57. The leukaemia-specific BCR–ABL transcript is an excellent target for molecular monitoring by quantitative PCR. There is no need for patient-specific primers because nearly all CML patients have one of two transcripts types (some patients have both) which differ by just one BCR exon.
The early quantitative PCR studies used titration assays which incorporated competitive targets49, 50, 51, 52, 53, 54, 55. Internal controls were introduced for some methods to control for the variation in the quality of the samples. The BCR–ABL copy number was normalised to the number of the control gene transcripts and the value expressed as a percentage ratio70. The technique was used to monitor patients after bone marrow transplantation where rising levels preceded cytogenetic and haematologic relapse and low or decreasing levels predicted remission50, 51, 71. It was also used as a sensitive indicator of response to donor leucocyte infusion following relapse after bone marrow transplantation72. Almost all patients treated with interferon alpha were shown to have detectable BCR–ABL transcripts70, 73. The terms PCR relapse or PCR progression were proposed to identify patients with pending relapse. The definition was a greater than 10-fold rise in BCR–ABL level in consecutive analyses, which reflected the inaccuracy inherent in the competitive PCR technique66.
Despite the apparent value of the competitive PCR assays for monitoring treatment response, its use was limited to specialised laboratories. Thoughtful experimental design and careful validation of competitive PCR techniques was absolutely critical for reliable results74. The technique was time consuming because multiple PCR reactions were required to titrate the sample with the competitor and extensive post PCR manipulation was necessary. Competitive PCR did not offer high throughput, precise quantitative analysis of BCR–ABL transcripts over a wide dynamic range.
The introduction of real-time PCR techniques in the late 1990s considerably simplified quantitative PCR analysis and have largely replaced the cumbersome competitive quantitative procedures. The kinetics of PCR are followed during amplification rather than at end-point which eliminates the need for co-amplification of a competitor. The fluorescence based technology enhances the reproducibility since quantitation is determined during the exponential phase of the PCR. The dynamic range is extended to over five orders of magnitude and real-time detection of the accumulating product eliminates the need for post PCR manipulation. Thus the possibility of PCR contamination is limited. The real-time technique is performed on an analyser that incorporates a thermal cycler, fluorescence detection and result calculation, which has greatly simplified quantitative PCR.
Nevertheless, the inherent technical complexities of quantitative PCR should not be overlooked and the successful application of real-time technology for reliable quantitation of BCR–ABL requires careful assay design and validation of all aspects of the procedure. For sensitive and reproducible results high quality RNA is essential. The appropriate selection of standards and control genes is of particular importance. It must be recognised that differences in the amplification efficiencies between DNA plasmid standards and the cDNA target can invalidate results75. Normalisation to the control gene compensates for variations in the quality of the RNA and for differences in the efficiency between reverse transcription reactions. The control gene must therefore degrade at the same rate as the target for accurate normalisation. The two control genes that have been widely assessed for BCR–ABL quantitation are BCR and ABL. Both are suitable in that they are expressed at low level and BCR has been shown to have a similar stability to BCR–ABL75. Appropriate design of primer and probe sequences are required to exclude the amplification of contaminating DNA and to avoid hybridisation at the polymorphic site in BCR exon 13. Primer binding in this nucleotide may falsely lower BCR–ABL transcript levels by up to 10-fold in patients with the polymorphism57, 75, 76. Rigorous precautions to exclude PCR contamination are still necessary when monitoring minimal residual disease.
The clinical usefulness of BCR–ABL quantitation by RQ-PCR has been demonstrated by several studies. RQ-PCR analysis of patients treated with imatinib has shown a strong correlation between the percentage of Ph-positive metaphases in the bone marrow and simultaneous study of peripheral blood BCR–ABL levels measured by RQ-PCR14, 15, 59, 75. Early reduction of BCR–ABL transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib and the reduction of BCR–ABL correlates with prognosis14, 59, 65.
The RQ-PCR methods vary in respect to the type of instrument used, the primer and probe location, the real-time chemistry and the control gene56, 58, 63, 68, 69, 77, 78. These differences can lead to a variation in the sensitivity and measurement reliability between methods. It is essential that each laboratory establish these limits for their method to allow accurate interpretation of serial monitoring. The estimation of measurement reliability and appropriate quality assurance according to international standards are important aspects of the development of any method used to monitor patients. However, for the measurement of BCR–ABL transcripts by quantitative PCR, certified international reference and control materials are currently not available. Several groups have been working towards standardisation between methods and are developing guidelines for data analysis and for the reporting of minimal residual disease79.
To demonstrate the effectiveness of imatinib and the value of RQ-PCR monitoring it is instructive to look at the results of the IRIS trial. This definitive study established a level of BCR–ABL that correlates with progression-free survival (PFS). In this trial, newly diagnosed patients with chronic phase CML were randomised to receive either interferon alpha plus low dose Ara-C (IFN-AraC) or imatinib 400 mg/day. Patients were allowed to cross over to the other arm if they lacked response, lost response or were intolerant to their first line therapy. The majority of patients on the IFN-AraC arm crossed over to imatinib within the first 18 months5. After 12 months of therapy, the majority of patients treated with first line imatinib achieved a complete cytogenetic remission (CCR, 0% Ph-positive cells in the marrow). This is approximately equivalent to a 2-log reduction in leukaemia load below a standardised baseline level as measured by RQ-PCR14. Patients who had achieved a CCR could not be further differentiated by cytogenetic analysis, nor could significant changes in leukaemia level be recognised until the time of cytogenetic relapse.
To further analyse the level of leukaemic reduction achieved it was necessary to measure the level of BCR–ABL by RQ-PCR in patients who had achieved CCR. We measured the number of BCR–ABL transcripts and normalised the value to the number of BCR transcripts by calculating a percentage ratio of BCR–ABL/BCR. These assays were performed in three laboratories internationally, Adelaide, Seattle and London. Due to differences in the methodologies of the three laboratories there were consistent differences in the BCR–ABL/BCR levels. To standardise the results all three laboratories analyzed the same 30 samples collected from CML patients prior to therapy. The median value of the 30 samples was calculated to indicate the median baseline value for that laboratory. All other results from patients on the study were then expressed as a log reduction from the median baseline value of the laboratory where tested. The advantage of defining molecular response according to reduction from a median pre-treatment level is that once a laboratory has established their median baseline level, results can be expressed on a common scale internationally. Another advantage is that molecular response can be calculated without needing to know the actual baseline level for that particular patient. For instance in one laboratory the median value for the thirty samples was a BCR–ABL/BCR value of 80%. A result of 0.8% in that laboratory can be expressed as a 2-log reduction below the standardised baseline without knowing the actual baseline value of the patient. This allowed all results to be expressed on a common scale so that results from the three laboratories could be pooled. In the molecular analysis of the IRIS study a “major molecular response” was defined as a ⩾3-log reduction in BCR–ABL/BCR level when compared to the median pre-treatment level.
Using this strategy, the level of log reduction achieved on the two arms of the study could be calculated and compared. After 12 months of therapy 93% of patients on the IFN-AraC arm had not achieved a CCR and were not subjected to RQ-PCR analysis. In the remaining 7% of patients, only 2% achieved a ⩾3-log reduction of BCR–ABL from the standardised baseline. By contrast, an estimated 39% of all first line imatinib treated patients achieved a ⩾3-log reduction by 12 months of therapy. A further 29% of imatinib treated patients achieved a CCR but they did not achieve a ⩾3-log reduction80.
The risk of progression according to the level of response achieved at 12 months showed there was a clear difference in PFS according to the level of leukaemia reduction. In this analysis the definition of progression included loss of a major cytogenetic response (MCR, less than 35% Ph-positive cells in the marrow), loss of a complete haematologic response, progression to accelerated phase or blast crisis and death from other causes while on imatinib. The risk of progression in patients who did not achieve a CCR by 12 months was 20%. In patients who achieved a CCR but not a 3-log reduction, the risk was 8%. However in the group who achieved a 3-log reduction at 12 months, not a single patient had disease progression with a 2-year follow up since the 12-month landmark analysis. We proposed that a 3-log reduction be defined as a major molecular response because it defined a group with remarkable stability of response, and represented a further 1 log reduction below the level of CCR80.
The terms “PCR negative” and “complete molecular response” should be used with caution. They imply an absolute lack of leukaemia, which may be misleading. There is inherent variability in the sensitivity of RQ-PCR and nested PCR assays between laboratories and between samples. Using current technology, sensitivity of >4.5 logs below baseline can be achieved with good quality RNA samples. For the IRIS study, a reduction in BCR–ABL level of ⩾4.5 logs was detected and verified on at least one occasion in 3.6% of patients who were in CCR (follow-up 18 months)80.
An RQ-PCR substudy to the IRIS trial was conducted in our laboratory in patients treated within Australia and New Zealand. The leukaemic load was measured in every patient at 3 or 6 month intervals, irrespective of their cytogenetic response. This study demonstrated that the level of BCR–ABL reduction was approximately 1 log by 12 months in the patients receiving first line IFN-AraC. Patients on first line imatinib achieved a median reduction in BCR–ABL level of over 2 logs by 6 months and over 3 logs by 18 months. Beyond 18 months there was a slow and steady fall in median values. At 30 months the median value was 4 logs below the baseline level. However even with a long follow up of 30 months, only 8% of patients had undetectable BCR–ABL. Interestingly those patients who crossed over from IFN-AraC to imatinib had a similar pattern of reduction in BCR–ABL to the pattern seen with first line imatinib patients14. These findings were confirmed in a similar study conducted in Germany15.
We found a good correlation between the BCR–ABL values and the percentage of Ph-positive cells in the marrow. Patients who did not achieve a MCR had BCR–ABL values clustered around the baseline value, whereas those who achieved a MCR but not a CCR had BCR–ABL values between 1 and 2 logs below the baseline in most cases. Almost all patients who achieved a CCR had BCR–ABL levels more than 2 logs below the baseline. Thus, a 1- and 2-log reduction by RQ-PCR is approximately equivalent to a MCR and a CCR, respectively. The correlation between blood RQ-PCR and marrow RQ-PCR is extremely high. When we compared these two values in a set of patients who had both studies performed simultaneously the correlation was r = 0.98. Marrow RQ-PCR does not seem to provide greater sensitivity to blood RQ-PCR in chronic phase CML14, 63.
The level of BCR–ABL reduction achieved early after commencing imatinib therapy is a good indicator of subsequent response14, 15, 59, 65. Based on the Australasian analysis of IRIS patients, those who do not achieve a 1-log reduction by 3 months have a very low probability of achieving a major molecular response (only 13% at 30 months). This compares to a probability of 100% and 69% in those achieving a >2-log and a 1–2-log reduction, respectively (Fig. 1). Failure to achieve a major molecular response in the group with a less than 1 log reduction at 3 months was largely due to the high level of primary and acquired resistance in this group. The estimated risk of resistance in this group was 83% compared to 5% and 0% in the 1–2 log and >2 log groups, respectively (Fig. 2).
One of the issues frequently raised when the concept of measuring log reduction is discussed is whether the individual patient baseline level is needed to determine the log reduction accurately. All of our analyses have been based on calculating the log reduction from a median baseline level rather than from the actual baseline level. However, when we reanalysed the predictive value of log reduction at 3 months according to the actual log reduction there was no significant difference in the response. Therefore, the actual log reduction did not provide a better prediction of subsequent response. In addition, we compared the median log reduction of BCR–ABL in patients with a high baseline value and those with a low baseline value in 123 patients who commenced imatinib as their first line of therapy. The median baseline value in these patients was a BCR–ABL/BCR% value of 113% and patients were divided into two groups based on this value. Those with a low baseline had a value of <113% and those with a high baseline had a value ⩾113%. For both groups, the median BCR–ABL/BCR% values at 3 monthly intervals were calculated and there was no difference in the median values from 3 months of imatinib (Table 1). This suggests that the baseline is not an important biological variable but more studies are needed before we can conclude that it has nothing to contribute to RQ-PCR analysis at the individual patient level.
Resistance can be broadly categorised as primary or acquired. Primary resistance is the failure to achieve (1) a complete hematologic response by 3 months or (2) MCR by 6 months. It is not known at this stage what causes primary resistance but it is rarely if ever caused by point mutations in BCR–ABL. By contrast point mutations are the cause of 35–90% of cases of acquired resistance. Acquired resistance can be defined as progression to blast phase, progression to accelerated phase, loss of haematologic response, loss of MCR or loss of CCR with a 10-fold rise in BCR–ABL81.
In the IRIS study, 4% of previously untreated patients did not achieve a complete hematologic remission on imatinib 400 mg/day. Failure to achieve a MCR by 6 months occurred in 23% of newly diagnosed patients with continuing imatinib at 400 mg/day. Thus, overall around 20–25% of newly diagnosed patients appear primarily resistant to imatinib at 400 mg/day.
In vitro preclinical studies were conducted by several groups to define the possible mechanisms of imatinib resistance82, 83, 84. They generated imatinib resistant cell lines that expressed BCR–ABL by exposure to gradually increasing levels of imatinib over several months. Multiple mechanisms of resistance were observed with the most frequent being overexpression of BCR–ABL mRNA and protein that was mediated through gene amplification or increased transcription. Other studies suggested that resistance may be associated with BCR–ABL independent mechanisms and mediated through overexpression of other kinases85, 86. The study of imatinib resistance in CML patient cells at the time of relapse has suggested that reactivation of BCR–ABL signalling plays a functional role at relapse with imatinib resistance and that kinase activity is critical for leukomogenesis27. Indeed, mutations in the BCR–ABL kinase domain that interfere with imatinib binding and lead to the reactivation of kinase activity are the most common mechanism of acquired resistance and more than 35 mutations have now been described27, 30, 31, 32, 33, 34, 37, 38, 44, 87, 88, 89. Additionally, amplification of the BCR–ABL gene can occasionally be detected in resistant patients44, 46, 87.
Monitoring imatinib-treated patients for BCR–ABL kinase domain mutations provides an essential guide for clinical management. However, monitoring all patients for mutations at regular time-points is not feasible in most centres. We have recently demonstrated that the emergence of a mutant clone is highly associated with a rising level of BCR–ABL as measured by RQ-PCR89. Even very small rises of just over 2-fold measured by a highly reproducible quantitative assay had biological significance in most cases tested, identifying patients with mutations. Therefore, patients with stable or decreasing BCR–ABL levels may not require mutation screening.
Patients with resistance due to mutations may need differing treatment strategies to manage resistance. Biochemical and cellular assays have demonstrated different levels of resistance for the various mutations30, 33, 87, 90, 91 and clinical studies have shown that mutations located within the region of the P-loop are associated with a poorer prognosis32, 37. Direct sequencing of the BCR–ABL kinase domain will reveal emerging mutant clones once they represent more than 10–20% of the leukaemic clones.
Nearly all mutations that become detectable by direct sequencing are associated with some evidence of acquired resistance32. In some patients, resistance develops rapidly and leads to a complete loss of the effect of imatinib. In other patients the mutant clone is still responsive to imatinib and the use of higher doses can re-establish full or partial control. The variable patterns of response are shown in Fig. 3. In our studies, the most common pattern of mutations when first detected is a mix of mutant and wild type BCR–ABL cells and the mutant may predominate with continued imatinib therapy (Fig. 4).
For valid and sensitive mutation results it is imperative that careful PCR design be undertaken. The segment of the BCR–ABL kinase domain that is located beyond the activation loop should be included in the sequencing analysis. In our studies, mutations are located in this region in 16% of patients with mutations. For direct sequencing a mutant sensitivity of 10–20% is dependent on PCR isolation of the BCR–ABL allele. This necessitates a PCR fragment design that includes a forward primer hybridising in BCR exon 13 (b2) and a reverse primer hybridising in ABL exon 9 or 10. A nested PCR strategy is required to amplify the kinase domain of patients with low BCR–ABL levels. For valid mutation data it is imperative that good quality RNA is analysed because poor quality samples may produce inconsistent mutation results (Fig. 5)32, 92. For this reason, we confirm the quality of the RNA by determining adequate transcript levels of a control gene using RQ-PCR32. The control gene level must be above a specific cut-off value before analysis is performed and different cut-off levels are used depending on the BCR–ABL transcript level. For samples with very low levels the RNA quality must be especially high to ensure amplification of the BCR–ABL allele in the semi-nested PCR and to produce valid mutation results. When a mutation is detected in a patient for the first time the mutation analysis is repeated to confirm its presence and to exclude an artefact.
The accuracy of mutation detection is also dependent on the efficiency of the PCR reaction. The amplification of quality control samples at appropriate BCR–ABL transcript level in the first and second round PCR can monitor for adequate efficiency92. We have observed unreliable sequencing results when the analysis proceeds despite failure of the control sample to amplify in the first round PCR. By ensuring that high quality RNA is analysed and by confirming the efficiency of the PCR, samples with BCR–ABL levels that correspond to approximately a 3.5-log reduction from the baseline can be screened successfully for mutations.
We have surveyed over 200 imatinib-treated patients regardless of whether response was maintained or lost. Three main groups were included; patients who commenced imatinib in the accelerated phase, chronic phase patients who commenced imatinib 12 months or more since diagnosis (late chronic phase), and patients who commenced imatinib in early chronic phase93. In all cases mutation screening using a direct sequencing strategy was performed at least 6 monthly or more often when resistance was indicated. There was a significantly higher probability of mutations according to disease phase with accelerated phase patients having a higher incidence than late chronic phase patients, and late chronic phase patients having a higher incidence than early chronic phase patients. In addition, within each disease phase the early cytogenetic response stratified the risk of detecting mutations with a higher probability in patients without a MCR by 3 months. Accelerated phase patients without a MCR had a particularly high risk of mutations that was estimated to be almost 80% by 24 months of imatinib therapy. Conversely, amongst patients who commenced imatinib in early chronic phase and achieved a MCR, there was a very low incidence of mutations. Within the patient categories there was a significantly higher probability of survival for patients without detectable mutations at 24 months of imatinib therapy93.
The data supports the concept that the leukaemic clone accumulates sequence errors during DNA replication, some of which affect BCR–ABL. The probability of developing subclones that are imatinib resistant would relate to the duration of disease and the size of the stem cell pool at risk. Gradually, a pool of BCR–ABL mutants would be generated which, if they have a lower affinity for imatinib, would selectively expand in the setting of imatinib treatment.
Section snippets
Conclusions
Based on the accuracy and sensitivity of RQ-PCR for measuring BCR–ABL levels in peripheral blood, this should become the method of choice for monitoring patients on imatinib and will facilitate comparisons of different imatinib-based treatment strategies. At the individual patient level, RQ-PCR studies can identify degrees of molecular response that predict long-term stability, as well as patterns of response that provide an early indication of relapse and imatinib resistance. For patients with
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