Elsevier

Leukemia Research

Volume 24, Issue 11, November 2000, Pages 951-956
Leukemia Research

Real-time quantitative reverse transcription-polymerase chain reaction for the detection of AML1-MTG8 fusion transcripts in t(8;21)-positive acute myelogenous leukemia

https://doi.org/10.1016/S0145-2126(00)00071-0Get rights and content

Abstract

Quantification of AML1-MTG8 fusion transcripts was performed by using real-time reverse transcription-polymerase chain reaction (RT-PCR) and the clinical value of this method was evaluated in t(8;21)-positive acute myelogenous leukemia (AML). A t(8;21)-positive cell line, Kasumi-1, was used for constructing standard curves and the corrected AML1-MTG8 mRNA expression level relative to the expression of the GAPDH housekeeping gene was calculated. Bone marrow samples from 14 patients with t(8;21)-positive AML were sequentially examined. The corrected AML1-MTG8 expression level at diagnosis varied in the range from 0.4 to 2.7 (median, 1.5) among the patients. When samples at 1, 3 and 6 months were examined after diagnosis, the corrected AML1-MTG8 expression level was found to decrease sequentially in all but one. AML1-MTG8 fusion transcripts were also detected in four of eight samples from patients in remission for more than 1 year. In conclusion, real-time RT-PCR can provide a rapid and accurate quantification of AML1-MTG8 fusion transcripts. This system could be useful to reveal the prognostic relevance of minimal residual disease in t(8;21)-positive AML.

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.

Section snippets

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,

References (19)

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