The interpretation of test results is influenced by the clinical context in which the test is performed. In the diagnostic setting, a negative RT-PCR result renders a diagnosis of CML unlikely. However, it does not definitively exclude a diagnosis of CML since rare variant breakpoints can occur that may not be detected by conventional PCR primer sets. Similarly, a positive qualitative RT-PCR result does not definitively invoke a diagnosis of CML. Using an extremely sensitive nested RT-PCR technique, BCR-ABL1 fusion transcripts have been identified in the blood of as many as two thirds of healthy adults.35,36 Whereas the vast majority of CML patients express either the e13a2 (b2a2) or the e14a2 (b3a2) fusion transcripts, healthy individuals express primarily the e1a2 type. However, the extreme level of sensitivity achieved (~10-8) with this nested technique is neither required nor recommended for routine clinical testing, and thus misdiagnosis should not be an issue.
For therapeutic monitoring, a single positive qualitative RT-PCR result is not predictive of relapse in any individual patient regardless of therapeutic context; nonetheless,ther-apeutic context ultimately dictates the clinical ramifications of any test result. For instance,in the setting of bone marrow transplantation, the interval after transplant, the type of transplant (i.e., unrelated vs related matched),and the presence or absence of stem cell product manipulation (e.g., T-cell depletion) may impact the prognostic relevance of qualitative test results. Most CML patients have a positive qualitative RT-PCR result in the first 6 months following ASCT that is not associated with a higher risk of relapse. Six to 12 months after ASCT, however, positive qualitative results are highly associated with, and considered to be an independent predictor of, subsequent relapse.37 In this setting, positive RT-PCR detection of BCR-ABL1 fusion transcripts precedes cytogenetic and hematologic relapse by several months. Furthermore, detection of fusion transcripts by RT-PCR 6 to 12 months after transplant with an allogeneic related matched donor or with T-cell-depleted stem cell products is associated with a higher rate of relapse than in patients receiving unrelated donor or nondepleted stem cell products.38
Despite attainment of complete cytogenetic remission, patients treated with interferon-a can continue to be qualitatively RT-PCR positive for several years. Furthermore, in the small number of patients treated with interferon-a who do become RT-PCR negative, FISH analysis can remain positive for the BCR-ABL1 gene fusion, supporting the notion that interferon-a may decrease chimeric gene transcription without successful eradication of the neoplastic clone.39 Consequently, qualitative RT-PCR has traditionally been of little clinical utility in the posttherapeutic monitoring of patients treated with interferon-a, while FISH analysis has provided more meaningful clinical data. A long-term outcome study (with an approximately 10-year follow-up), however, has documented that repeated negative qualitative RT-PCR tests, or even the attainment of at least one documented molecular remission after inter-feron-a therapy, is associated with improved long-term event-free survival as well as the prolonged durability of a major cytogenetic response.19 Although this finding offers validity to qualitative RT-PCR monitoring during or after interferon-a therapy, it is relevant to few CML patients.
In contrast to qualitative RT-PCR analysis, quantitative RT-PCR shows seemingly broad relevance to postthera-peutic monitoring whether after transplantation, or after therapy with interferon-a or imatinib mesylate. Low or falling transcript levels correlate with successful treatment or continued remission, and elevated or rising transcript levels predict relapse.14,16,17 The establishment of specific criteria, critical thresholds to define molecular relapse, or both has, however, been difficult in light of the ongoing evolution and continued sophistication of quantitative RT-PCR technology. The European Investigators on CML (EICML) issued recommendations in 1994 that defined a quantitative RT-PCR relapse as a 10-fold or greater increase in the level of BCR-ABL1 transcripts, as determined by a minimum of three consecutive quantitative analyses.40 Although these guidelines offer a framework for monitoring, they were developed over a decade ago and prior to the advent of real-time RT-PCR technology.
Since then, much data have been generated though still compromised by the absence of large prospective clinical trials and the lack of technical standardization; nevertheless, pertinent information can be ascertained. With respect to monitoring after transplantation, several quantitative indicators emerge. Thus, a modified definition of molecular relapse has been proposed as rising or persistently high levels of BCR-ABL1, delineated as a BCR-ABL1/ABL1 ratio of >0.02% (~10-4),in two sequential specimens procured more than 4 months after ASCT. Although imatinib has shown encouraging results in the treatment of CML,the long-term correlates of clinical outcome remain to be defined. Nonetheless, recent studies suggest that quantitative RT-PCR values correlate with established cytogenetic response criteria and are associated with a differential short-term outcome.41 In the IRIS study, a "major molecular response (MMR)" is defined as a >3 log reduction in BCR-ABL1/BCR levels compared to median pretreatment levels. Here, MMR was achieved in 39% of newly diagnosed CML patients after 12 months of imatinib as compared to only 2% of patients on interferon-a plus Ara-C.17 Patients with this degree of molecular response to imatinib had a negligible risk of disease progression in the short term. In addition, the patterns of residual disease levels after attaining MMR (such as continuing decline in levels, plateau, or increasing levels) can predict cytogenetic relapse.42
One additional advantage of quantitative BCR/ABL1 monitoring with imatinib therapy is that it allows for the early recognition of therapeutic resistance. In this scenario, patients with a suboptimal therapeutic response may undergo screening for causative point mutations (e.g., using allele-specific oligonucleotide PCR, denaturing HPLC, or heteroduplex formation), the identification of which could prompt implementation of alternative treatment strategies. Preliminary work with microarrays has identified differential gene expression profiles between patients with imatinib sensitivity and resistance.43 In the future, this technology may predict therapeutic response and thus dictate frontline therapy.
Regardless of the therapeutic strategy, large prospective studies that correlate quantitative RT-PCR data with long-term clinical outcome are anticipated. These investigations
BCR-ABL1/ABL1 > 0.02% 2 sequential speciments 4 months post-ASCT
Below Q-PCR detection I
Below Q-PCR detection I
negative x 1 yr negative x 1 yr
Interferon-a f +
> 3 log reduction BCR-ABL l/ABLl
> 3 log reduction BCR-ABL l/ABLl
Major molecular response
Imatinib mesylate i i
>3 log reduction BCR-ABLl/ABLl I Major molecular response j
>1 log increase BCR-ABL1/ABL1
[ Acquired resistance |
>3 log reduction BCR-ABLl/ABLl I Major molecular response j a b c
Figure 35-7. Algorithms for the frequency of therapeutic monitoring in CML: (a) after allogenic stem cell transplantation (ASCT), (b) with interferon-a therapy, and (c) with imatinib mesylate therapy. FISH, fluorescence in situ hybridization; mos, months; Ph, Philadelphia chromosome; RT-PCR, qualitative reverse transcription polymerase chain reaction; Q-PCR, quantitative reverse transcription polymerase chain reaction; wk(s), week(s). Recent recommendations propose that FISH is no longer an appropriate method for monitoring patients in complete cytogenetic remission.78 <-
will direct the formulation of new guidelines to assist with the future therapeutic monitoring and clinical interpretation of these tests. Until then, a number of algorithms have been proposed for therapeutic monitoring25,26,44,45 (Figure 35-7).
One final caveat in molecular RT-PCR analysis, whether qualitative or quantitative,is that it cannot assess the clonal evolution of disease. For this assessment, cytogenetic karyotyping is required and must be performed every 6 months throughout the monitoring process regardless of the type of therapy. The emergence of cytogenetically abnormal (but Ph-negative) clones with the use of imatinib, with a reported incidence ranging between 2% and 24%, further underscores the need for periodic conventional cytoge-netic analysis.46
Was this article helpful?