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PCR Testing: Setting the Standards for the Future of Oncology
This page provides information for understanding the role of molecular response assessment for optimizing targeted therapy for patients with Ph+ CML. More than 95% of patients with CML exhibit the Ph chromosome, which is generated by a balanced reciprocal translocation between the long arms of chromosomes 9 and 22, namely the t(9;22)(q34;q21).1 This aberrant chromosome encodes a fusion gene, Bcr-Abl. Depending on the position of the Bcr breakpoint, various Bcr-Abl fusion gene transcripts are translated into functional Bcr-Abl proteins (p190, p210, and p230). Most common of these are the 210-kD proteins, usually referred to as p210 Bcr-Abl. Constitutive activity of the Abl tyrosine kinase portion of the p210 Bcr-Abl fusion proteins has been shown to be critical in the pathogenesis of Ph+ CML.1 Response Assessment in CMLResponse to Ph+ CML therapy is monitored by three different methods with increasing sensitivity for detection of residual disease. (Figure 1) Hematologic response is assessed by peripheral blood cell counts and by spleen size; cytogenetic response is assessed by determining the percentage of the Ph-chromosome positive metaphases in the bone marrow, and molecular response is assessed by measuring levels of Bcr-Abl gene transcripts.
Quantitative detection of Bcr-Abl transcripts to determine molecular response was made commonly feasible by the introduction of “real-time” quantitative polymerase chain reaction methodology (RQ-PCR).3-6 Bcr-Abl transcript levels measured in both bone marrow and peripheral blood samples from Ph+ CML patients have been shown to correlate with the number of the residual leukemic cells in the sample as established by cytogenetic analysis. (Figure 2) RQ-PCR can also be very sensitive as it can detect a single Bcr-Abl –positive cell among 105-106 normal cells, although the most commonly reached sensitivity is in the range of 104-105.
Molecular ResponseThe value of molecular response assessment in evaluating therapeutic efficacy for Ph+ CML therapy was demonstrated in the phase 3 International Randomized Study of Interferon plus Ara-C and STI571 (IRIS) trial.2 Ph+ CML patients in the IRIS trial treated first-line with targeted therapy achieved significantly higher overall rates of complete hematologic and cytogenetic responses compared with the combination (interferon) therapy.2 The degree of efficacy was further explored by measuring Bcr-Abl transcripts to assess molecular response and the residual disease burden in patients who achieved a complete cytogenetic remission.3 Molecular responses were evaluated using RQ-PCR methodology.3 Bcr-Abl transcript levels were expressed as a percentage ratio of Bcr-Abl to BCR transcripts. Bcr served as a control to compensate for variations in the quality of the RNA and for differences in the efficiency of the reverse transcription reaction. To standardize the results generated in the three testing laboratories, a pretreatment baseline value was established. Bcr-Abl transcripts were measured in blood samples from 30 patients with newly diagnosed Ph+ CML-CP prior to treatment in each of the testing laboratories. The median Bcr-Abl/Bcr percent value in each laboratory was designated as the standard baseline for that laboratory. A reduction in Bcr-Abl transcript levels of at least a 3 log (1,000-fold) reduction below a standardized baseline derived from a median ratio of Bcr-Abl to Bcr was obtained from 30 untreated patients with chronic-phase CML who participated in IRIS. Thus, molecular responses were reductions from an absolute baseline (common to all) rather than a relative baseline (individualized). This ensures that patients with the same level of molecular response have the same degree of residual disease. Moreover, under- or overestimation of the extent of molecular response due to individual variations in pretreatment disease levels is avoided by using a common standard baseline. The IRIS study avoided the term complete molecular response (CMR) but rather determined the number of patients with undetectable Bcr-Abl with a sensitivity of 4.5 log below the standardized baseline.8 Recognizing that this level of molecular response reflects the limits of current detection methods, undetectable Bcr-Abl should not be equated with eradication of Bcr-Abl L expression or cure. Cases in which patients with undetectable Bcr-Abl discontinued targeted therapy and then subsequently relapsed reinforce that the lack of detection of Bcr-Abl transcripts is neither a cure nor a reason to discontinue targeted therapy.4-6 Molecular Response Correlates with Cytogenetic ResponseIn the IRIS trial, patient blood samples were monitored for Bcr-Abl transcript levels within 2 weeks after achieving complete cytogenetic response (CCyR) and then every 3 months.3 Patients treated first-line with Glivec® therapy had significantly higher rates of both cytogenetic and molecular responses compared with patients treated with combination (interferon) therapy, suggesting that cytogenetic responses correlate with molecular responses. Molecular Response Predicts Progression-Free SurvivalCytogenetic responses are an established prognostic indicator of progression-free survival in CML.7,8 Furthermore, long-term analysis of IRIS data also supports for the first time the prognostic significance of molecular responses in Ph+ CML therapy.9 Overall, CML patients with CCyR and MMR at 12 months on first-line targeted therapy had the lowest rate of progression (zero) to accelerated phase (AP) or blast crisis (BC) at 60 months compared with patients with CCyR but without MMR. It is noteworthy, however, that at 18 months patients with CCyR have more stable responses and a significantly higher rate of freedom from progression to AP/BC (98%-100%) with respect to patients without CCyR (87%). Consensus Recommendations for ResponseAssessment In Ph+ CMLEvidence obtained in clinical trials and post-approval setting with targeted therapy has prompted experts to formulate consensus recommendations for response assessment and treatment of patients with Ph+ CML.10-13 The European LeukemiaNet recommendations propose a schedule of response expectations at various time points of targeted therapy. (Figure 3)
Molecular Analysis at DiagnosisAssessment of Bcr-Abl transcript, by RQ-PCR at diagnosis is necessary to confirm Ph+CML and establish the type of Bcr-Abl fusion present.4 In rare, complex karyotypic rearrangements, fluorescence in situ hybridization (FISH) must be performed to detect the presence of gene deletions or variant translocations, whose precise prognostic significance still needs to be established. Molecular Monitoring During TherapyEuropean LeukemiaNet experts recommend continued molecular monitoring during targeted therapy. Patients with Ph+ CML should have molecular response assessment performed at regular 3-month intervals.10,13 A steady decline in Bcr-Abl transcripts indicates an ideal response to therapy. The IRIS trial demonstrated that molecular responses generally continue to improve with time on targeted therapy. A substantial portion of patients without CCyR or MMR at 12 months, and some after 18 months, eventually achieved these levels of molecular response during continued therapy.15 In a recent study, a number of patients without CCyR at 1 year could reach the same levels of molecular response as those patients with CCyR at 12 months after 3 or 4 years.16 Together these results indicate that the time at which patients may achieve molecular responses varies and some patients require a year or more to achieve MMR. Ph+ CML patients who achieve CCyR and MMR have the best prognosis compared with patients who achieve only CCyR without MMR or no CCyR.9 A 5-fold to 10-fold (0.5 or 1 log) increase in Bcr-Abl transcript levels is currently proposed as the threshold for molecular relapse or resistance.12 This avoids the potential for raising concern over potentially clinically irrelevant fluctuations in Bcr-Abl transcript levels. Such fluctuations, however, indicate that the measurement reliability of the RQ-PCR assay should be optimized. A rise in Bcr-Abl transcript levels can be used to trigger analysis for resistant Bcr-Abl mutations. At present, direct gene sequencing for mutation detection is a commonly used approach. Finally, it must be considered that a meaningful rise in Bcr-Abl transcripts could also be due to inadequate drug levels caused by pharmacokinetic factors or lack of adherence to therapy. Molecular Monitoring MethodologyThe Ph+ CML community has recognized a need to harmonize methodology for detecting and reporting Bcr-Abl transcript levels. To that end, a consensus meeting of leading investigators in Ph+ CML was held in October 2005 at The National Institutes of Health (NIH) to harmonize RQ-PCR methodology worldwide.17,18 The discussion included key suggestions for stabilizing RNA samples, selection of a suitable control gene, as well as optimizing the methodology. Another important consideration discussed at the NIH meeting was the need for a Bcr-Abl standard for quantitation. Bcr-Abl transcript levels in the IRIS trial were plotted with respect to a standard baseline derived from the median value obtained from samples from 30 patients with newly diagnosed Ph+ CML-CP.3 Unfortunately, the patient samples used in the IRIS trial to establish the standard baseline are no longer available. The advent of commercially prepared Bcr-Abl standards is likely to address this problem. Recommendations for uniform expression of molecular response data were also formulated at this meeting.17 An International Scale for expressing molecular response in Ph+ CML, based on absolute values, was proposed.17,18 To harmonize the scale across laboratories worldwide, Bcr-Abl/control gene transcript ratios obtained in separate laboratories and known to correspond to a 3-log reduction in Bcr-Abl transcripts from the IRIS standard baseline will each be converted to 0.10% by arithmetic conversion factors. The International Scale will therefore be anchored to a critical value with known prognostic significance.19 Uniform expression of molecular monitoring data is anticipated to facilitate the understanding and use of this information for clinicians and scientists worldwide. SummaryMolecular monitoring is clearly an important and sensitive tool for evaluating responses as well as for predicting progression-free survival or relapse during targeted therapy for Ph+ CML. Although peripheral blood sampling is relatively easy to perform, obtaining reliable molecular monitoring data can be a challenge for clinicians not associated with institutions routinely performing these assays for molecular response. Lack of access to or unreliable molecular response data can compromise therapy for patients with Ph+ CML. Reliable and timely molecular monitoring during therapy has the potential to guide clinical decision making toward optimizing targeted therapy for Ph+ CML. References
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