Real-time quantitative reverse transcription-polymerase chain reaction for the detection of AML1-MTG8 fusion transcripts in t(8;21)-positive acute myelogenous leukemia
Introduction
In the management of patients with acute leukemia, complete remission (CR) is usually determined by morphologic assessment. However, patients achieving morphologic CR may still have as many as 1010 leukemic cells [1], and some of them subsequently relapse. Identifying much lower levels of residual disease may help predict the risk of relapse, thus contributing to improving the treatment strategies.
Minimal residual disease (MRD) in acute leukemia has been detected by several techniques, including conventional cytogenetic techniques, cell culture techniques, immunologic methods, and polymerase chain reaction (PCR) [1], [2]. However, techniques other than PCR are not so advantageous with respect to sensitivity and are not useful for lowering the threshold of disease detection. On the other hand, the sensitivity of PCR for the detection of MRD is extremely high. However, the PCR method is usually used for qualitative assessment.
Recently, a real-time quantitative PCR method based on the use of the 5′ nuclease assay first described by Holland et al. [3], was described as providing a very accurate and reproducible quantification of gene copies [4], [5]. Briefly, during the extension phase of PCR, the 5′ nuclease activity of Taq DNA polymerase cleaves dual-labeled fluorogenic hybridization probes (TaqMan probes) which are designed to hybridize internally to the PCR primers. The cleavage of the probe results in an increase of the reporter dye fluorescence proportional to the number of target genes and it is monitored in real time during PCR amplification. This quantitative PCR assay has been used to detect and quantify MRD in some diseases [6], [7], [8], [9].
In this study, we performed real-time reverse transcription-PCR (RT-PCR) for the detection of AML1-MTG8 fusion transcript in acute myelogenous leukemia (AML) with t(8;21) in order to test whether this system provides an accurate quantification of MRD.
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Patients and samples
In this study, we used the cell line Kasumi-1 established from a patient with t(8;21)-positive AML [10] as a positive control.
A total of 14 patients with t(8;21)-positive AML, whose bone marrow (BM) samples were available at diagnosis and at several interval points following achievement of remission, were included in this study (Table 1). Although the treatment schedule was not uniform, all patients had achieved CR after induction chemotherapy including cytarabine, mitoxantrone and etoposide.
Amplification curves and standard curves
Amplification curves of serially (1:10) diluted Kasumi-1 RNA samples amplified with AML1-MTG8 primers are shown in Fig. 1A, in which the changes in the fluorescence signal of the reporter dye (ΔRn) are plotted on the y-axis and cycle number is plotted on the x-axis. The amplification plots shifted to the right as input RNA was reduced. In this system the threshold cycle (CT), defined as the cycle number at which the ΔRn of a sample crossed a threshold of 10 S.D. above the baseline fluorescence,
Discussion
The threshold of MRD detection for patients with leukemia has been lowered by the development of PCR methods. In patients with t(8;21) AML, the detection of AML1-MTG8 fusion transcript using qualitative RT-PCR has been reported in several studies [11], [12], [13], [14], [15]. However, it is not always useful for monitoring MRD because AML1-MTG8 fusion transcripts are detectable in patients in long-term remission after treatment [2].
In order to reveal the value of MRD in the clinical management
Acknowledgements
M Kondo provided the concept, design, drafted the paper, gave statistical expertise, helped to collect data and provided funding. K Kudo and H Kimura contributed to the concept and design, provided technical support and helped with data interpretation. J Inaba and K Kato provided study materials and assembled the data. S Kokima and T Matsuyama provided study materials. K Horibe contributed to the concept and design, gave statistical expertise, administrative support, provided critical revision,
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