1Molecular Diagnostics Laboratory, 3Cytogenetic Laboratory, Division of Pathology and Laboratory Medicine, 2Department of Leukemia, 4Department of Immunology and Biological Therapy, Division of Medicine, The University of Texas MD Anderson Cancer Center, Houston, USA
Received 2 May 2001; revised 13 November 2001; accepted 11 December 2001.
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Abstract |
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Quantitative real-time polymerase chain reaction (Q-Rt-PCR) is a new tool in the detection and quantification of the BCR/abl fusion transcripts in chronic myelogenous leukemia (CML). This study investigates its specificity, sensitivity and potential clinical usefulness.
Patients and methods
Parallel analysis of Q-Rt-PCR and the conventional reverse transcription-mediated PCR (RTPCR) were performed on 567 samples from 481 patients. Treatment response was monitored by Q-Rt-PCR at 6 and 12 months of 61 patients on STI-571 and 103 patients on interferon.
Results
The concordance rate between Q-Rt-PCR and RTPCR was 96.3% (546/567), with 341 positives and 205 negatives. The positive equivalents ranged from 2 x 106 to 1.2 µg of K562 cell RNA. Karyotyping in 372 samples revealed excellent correlation with Q-Rt-PCR measurements (P <0.001). Setting residual BCR/abl <0.01 as an early goal of molecular response, we observed that STI-571 induced a better response than interferon: 49% (20 of 41 patients) versus 35% (15 of 62 patients) at 6 months (P = 0.025) and 52% (32 of 61 patients) versus 34% (35 of 103 patients) at 12 months (P = 0.01), respectively.
Conclusions
Q-Rt-PCR provides reliable measurements of BCR/abl fusion transcripts. It is potentially useful in assessing molecular residual disease after therapy.
Key words: chronic myelogenous leukemia, interferon, real-time polymerase chain reaction, STI-571
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Introduction |
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Recently, PCR technology has evolved into real-time fluorescence detection. One of the detection methods takes advantage of the 5'3' exonuclease activity of Taq DNA polymerase, which hydrolyzes a double-labeled fluorogenic probe upon annealing to the target PCR products during the PCR sequence extension phase [6]. Since fluorescence acquisition is monitored cycle-by-cycle, product analysis can be performed while PCR is in progress. It provides not only detection but also quantification of the accumulated target PCR products. To validate quantitative real-time PCR (Q-Rt-PCR) in the detection of BCR/abl fusion transcripts, we performed side-by-side comparison analysis with conventional reverse transcription-mediated PCR (RTPCR). To determine the reliability of Q-Rt-PCR in quantification, the measurements of BCR/abl fusion transcripts were correlated with the results of cytogenetic analysis. To explore its potential clinical usefulness, we investigated the use of Q-Rt-PCR in assessing the molecular residual disease after therapy in Ph-positive CML patients. We were particularly interested in comparing interferon-based therapy [7] with a new treatment, STI-571 (a potent and specific tyrosine kinase inhibitor), which demonstrated promising therapeutic efficacy in recent clinical trials [810].
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Patients and methods |
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Q-Rt-PCR assay for the detection and quantification of the BCR/abl fusion transcripts
RNA was extracted from bone marrow samples using Trizol reagent (Gibco-BRL, Gaithersburg, MD, USA) according to the manufacturers instructions. The integrity of the RNAs was determined by gel electrophoresis followed by ethidium bromide staining. Samples with intact 28S and 18S RNAs were considered adequate and were subjected to reverse transcription, which was performed on 1 µg of total RNA using random hexamers and superscript II reverse transcriptase (Gibco-BRL), as recommended by the manufacturer. The resulting cDNA was subjected to a multiplex PCR to co-amplify three different types of BCR/abl fusion transcripts, b2/a2, b3/a2 and e1/a2, and an internal standard, retinoic acid receptor-alpha (RAR), using a modification of the methods described previously [11, 12] with the primers shown in Table 1. Also added to the PCR reactions were double-labeled fluorogenic probes specific for the c-abl gene and RAR
gene at 0.2 µM and 0.05 µM, respectively (Table 1). The c-abl probe was labeled with 6-carboxy-fluorescein (6-FAM) at the 5' end and 6-carboxytetramethyl rhodamine (TAMRA) at the 3' end. The RAR
was labeled with VIC at the 5' end and TAMRA at the 3' end. Amplification of the internal standard RAR
allowed us to normalize variations in the efficiencies of reverse transcription and PCR, and to verify the integrity of RNA samples.
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Cytogenetic analysis
Cytogenetic analysis was performed according to established procedures [13] on bone marrow samples. Karyotypes were read according to the guidelines of the International System for Human Cytogenetic Nomenclature. For most samples, 20 metaphases were examined. In some instances, 1530 metaphases were counted. Samples with <15 metaphases were considered insufficient yields and excluded from correlation with Q-Rt-PCR measurements.
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Results |
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Assessing molecular response by Q-Rt-PCR after interferon and STI treatments
To investigate the potential clinical usefulness of Q-Rt-PCR, we studied 164 Ph-positive CML patients who had been treated with interferon-based therapy (103 patients) or STI-571 (61 patients). The patients clinical states at the time treatment started varied. Patients receiving interferon were in the chronic phase of disease. In contrast, patients treated with STI-571 were either in the accelerated phase of CML or had cytogenetic abnormalities, suggesting clonal evolution. Arbitrarily defining residual BCR/ablnorm <0.01 as an early goal of molecular response after therapy, we studied the molecular response rates within the first year of treatment (Table 5). At 6 months, 20 of 41 patients (49%) on STI-571 had residual BCR/abl fusion transcripts <0.01, as compared with 15 of 62 patients (35%) on interferon (P = 0.025). At 12 months, 32 of 61 patients (52%) on STI-571 and 35 of 103 patients (34%) on interferon had residual BCR/abl fusion transcripts <0.01 (P = 0.01).
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Discussion |
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Employing both conventional RTPCR and Q-Rt-PCR, we performed parallel analysis of a large number of CML samples in various clinical states alongside 11 samples obtained from normal donors as negative controls. We observed a high concordance rate of 96.3%, indicating that the sensitivity and specificity of both assays are equivalent. Although there were rare discrepancies (3.7%) in this study, parallel comparison helped to investigate the causes and the patterns of false positives and false negatives in both assays. Particularly noteworthy was the characteristic false-positive pattern observed in Q-Rt-PCR: an early take-off amplification curve with a low plateau. This could be due to autohydrolysis of the fluorogenic probe as a result of repeated heating and cooling during PCR cycles, or other unknown mechanisms. These false-positive measurements were frequently high according to Q-Rt-PCR, but cytogenetic analysis and conventional RTPCR were negative.
Three samples showed a weak Q-Rt-PCR measurement at 2 x 104, 1.1 x 104 and 2.6 x 105, respectively. However, conventional RTPCR did not detect the abnormalities. These false negativities in conventional RTPCR were most likely due to suboptimal Southern transfer, probe hybridization or signal enhancement by chemiluminescence, because the repeated analyses by both Q-Rt-PCR and conventional RTPCR confirmed the positive results.
There were 12 weak positives shown by conventional RTPCR, but Q-Rt-PCR showed a small curve under the threshold level in seven samples and did not forming an amplification plot in the remaining five. In fact, 11 of these 12 false negatives in Q-Rt-PCR were in cytogenetic remission, where the number of BCR/abl fusion transcripts was very low.
To test the reliability of the Q-Rt-PCR assay in the quantification of the BCR/abl fusion transcripts, we also performed a parallel correlation test between Q-Rt-PCR and cytogenetic analysis. Although these two assays were independent, we observed a highly significant correlation in which large quantities of BCR/abl fusion transcripts were associated with a high percentage of Ph-positive cells in the samples (P <0.001). This observation indicates that Q-Rt-PCR provides reliable BCR/abl fusion transcript measurements in Ph-positive CML. However, there were a few exceptions. Large quantities of BCR/abl fusion transcripts, measuring >0.01, were observed in three samples with 0% Ph chromosome. Also, small quantities of BCR/abl fusion transcripts measuring <0.001 were observed in 13 samples with >91% Ph chromosome (Table 4). These discrepancies prompted us to review the clinical states of these patients. The first patient who had 0% Ph chromosome and a large quantity of BCR/abl transcripts (1.2 x 101) actually had blast crisis of CML, for which chemotherapy with HyperCVAD (hyper-fractionated combination of Cytoxan, Vincristin, Adriamycin and Dexamethasone) was given. The sample was obtained 1 month after the first cycle of chemotherapy. The second patient who showed 0% Ph chromosome and high BCR/abl transcripts (1.6 x 102) had disease progression from late chronic phase to accelerated phase, as evidenced by increased blasts. However, the third patient (BCR/ablnorm 1.0 x 102) was still in cytogenetic and clinical remission. We also reviewed the clinical history of the 13 patients who had >91% Ph chromosome with small amounts (<1 x 103) of BCR/abl fusion transcript. Six of these patients were on chemotherapy for accelerated phase or blast crisis. The remaining seven patients were on treatment for chronic phase CML. Although the biological and clinical significance of these unusually low or high expressers is still unclear, they are rare exceptions and do not affect the analysis of this study.
The excellent correlation between Q-Rt-PCR measurements and the results of cytogenetic analysis justified the use of Q-Rt-PCR determinations to define molecular responses after therapy. In this study, we were particularly interested in patients after interferon-based therapy and patients on STI-571 treatment. Patient results following allogeneic bone marrow/peripheral blood stem cell transplants were not presented; they will be analyzed in a separate study.
In a previous study at The University of Texas MD Anderson Cancer Center, Kantarjian et al. [20] reported that the major cytogenetic response rate of early chronic phase CML to a daily dose of interferon plus low dose ara-C combination was 50%, with a median time to achievement of 7 months. In our current study, most patients on IFN therapy were also in early chronic phase, but they received more intensive therapy, a combination of IFN-, ara-C and homoharrintonin. However, only 34% of our patients could attain a molecular response of <0.01 at 12 months. Therefore, the question is raised of how a molecular response of <0.01 compares with a major cytogenetic response of <35% of Ph chromosome. In this study, of the 195 samples containing
35% of Ph chromosome, 150 (77%) had BCR/abl fusion transcripts measured as
1 x 102. On the other hand, in the 177 samples with <35%, 156 (88%) had BCR/ablnorm measured as <1 x 102. Although it is still too early to draw a conclusion, these findings support the use of residual BCR/abl fusion transcripts <0.01 as the early goal of molecular response within the first year of interferon or STI-571 treatment.
Based on this criteria, we observed that the response rate of STI-571 treatment was better than interferon therapy: 49% versus 34% at 6 months and 52% versus 34% at 12 months (P = 0.025 and 0.01, respectively). Of note, patients receiving STI-571 treatment were in accelerated phase or clonal evolution as opposed to most patients on interferon therapy being in early chronic phase. The better molecular response rates observed in STI-571 treatment suggested the potential clinical usefulness of STI-571. Nonetheless, long-term clinical follow-up is needed to determine whether the early molecular response will eventually translate into durable therapeutic effectiveness.
Since Q-Rt-PCR had a much higher sensitivity than cytogenetic analysis, Q-Rt-PCR helped identify a subset of complete cytogenetic responders whose leukemia cell burden was extremely low or undetectable. Moreover, in those patients with Ph-positive cells detected by cytogenetic analysis, the Q-Rt-PCR measurements of the BCR/abl fusion transcripts correlated very well with the amounts of Ph-positive cells as determined by cytogenetic analysis. Therefore, Q-Rt-PCR reliably measures leukemia cell burden, ranging from full-blown disease to extremely small numbers and complete freedom of Ph-positive cells. More importantly, the quantitative assessment could provide unprecedented valuable information that could help predict treatment response, drug resistance and imminent disease recurrence. If the clinical significance and prognostic values of the Q-Rt-PCR assay are confirmed by other studies and multi-center collaboration, it will eventually become a gold standard for disease monitoring in Ph-positive CML.
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Acknowledgements |
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Footnotes |
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