Leukemia (2000) 14, 329–335  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu BIO-TECHNICAL METHODS SECTION (BTS) BTS Leukemia Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia MP Wattjes 1 , J Krauter 1 , S Nagel 1 , O Heidenreich 2 , A Ganser 1 and G Heil 1 1 Department of Hematology and Oncology, Hannover Medical School; and 2 Department of Molecular Biology, Institute of Cell Biology, University of Tu ¨ bingen, Germany The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leuke- mia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hem- atological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel com- petitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both tech- niques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages Leukemia (2000) 14, 329–335. Keywords: AML; competitive RT-PCR; minimal residual disease; real-time PCR Introduction The reciprocal translocation t(8;21)(q22;q22) is one of the most frequent chromosomal aberrations in acute myeloblastic leukemia (AML). 1,2 This aberration results in the fusion of two genes, AML1 on chromosome 21 and MTG8 on chromosome 8, leading to the AML1/MTG8 fusion gene on the derivative chromosome 8 and the transcription of a chimeric AML1/MTG8 fusion mRNA. 3,4 Since the breakpoints of this chromosomal aberration cluster within the same intron, 5 identical AML1/MTG8 fusion transcripts can be detected by RT-PCR in all patients with t(8;21). In t(8;21) positive AML patients usually a high complete remission rate can be achieved by a standard induction Correspondence: G Heil, Dept of Hematology and Oncology, Hann- over Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Ger- many; Fax: 49 511 532 3611 Received 6 July 1999; accepted 20 October 1999 chemotherapy while the long-term prognosis of these parti- cular patients still remains unclear. 6–9 The detection of AML1/MTG8 by RT-PCR has been used to monitor the leu- kemic burden during and after chemotherapy with the aim of identifying patients with a high risk of relapse. However, using qualitative RT-PCR, several groups have shown that the vast majority of t(8;21) patients treated with conventional chemo- therapy or even autologous or allogeneic stem cell transplan- tation consistently have detectable levels of AML1/MTG8 fusion transcripts thus questioning the prognostic value of these analyses. 10–16 Preliminary data indicate that quantitative RT-PCR techniques are suitable to determine the chemosensi- tivity of the leukemic clone and to predict a relapse in these patients. 17–19 The quantification strategy of competitive PCR is based on the co-amplification of an internal RNA or DNA standard molecule of different size or with an additional restriction site. 20 Though competitive RT-PCR is time con- sumptive and needs further processing after amplification, it is widely used and validated for the quantification of specific DNA or RNA targets in hematological and infectious diseases. 21–24 Recently, real time PCR, a new quantitative PCR method based on the 5 –3 exonuclease activity of the Taq DNA-poly- merase and a fluorescence energy transfer, was intro- duced. 25,26 This method is increasingly used for the quantifi- cation of fusion transcripts in a wide variety of hematological diseases. 27–31 In contrast to competitive PCR, this technique uses standard curves derived from the amplification of an external standard, which is carried out in separate PCR tubes, for quantification. 32,33 It is therefore not clear if results obtained by competitive or real-time PCR can be directly compared. To further analyze this problem, we compared the sensitivity, linearity and reproducibility of real-time PCR with that of an optimized nested competitive RT-PCR assay for the detection and quantification of AML1/MTG8 fusion transcripts. Patients and methods Cell lines The t(8;21) positive AML cell line Kasumi-1 34 and the promye- locytic cell line NB4 35 were obtained from the German Col-