Quantitative detection of AML1-ETO rearrangement by real-time RT-PCR usingfluorescently labeled probes

The persistence of the AML1-ETO rearrangement performed by reverse transcri ption polymerase chain reaction (RT-PCR) has been reported in acute myeloid leukemia (AML) patients in long-term complete remission (CR). This persist ence, which is not associated with hematological relapse, limits the clinic al use of qualitative RT-PCR. Here, we present a new quantitative real-time PCR method to detect AML1-ETO rearrangement using fluorescently labeled pr obes. Quantitative detection of AML1-ETO was performed in capillary tubes u sing two fluorescently labeled probes in the LightCycler equipment. The rel iability of the method was checked in twenty-two bone marrow samples and on e apheresis sample from eight patients with t(8;21) collected at diagnosis and during follow-up assessment. The regression coefficients obtained for s tandard curves of AML1-ETO and AML were all greater than 0.98. The sensitiv ity attained allowed the detection of rearrangements at a dilution of 10(-5 ) Kasumi-1 cDNA. The intra-assay coefficient of variation was 4% for AML1-E TO, and 7% for AML. The inter-assay coefficient of variation was 19% for AM L1-ETO and 12% for AML. A log reduction from two to four in the AML1-ETO/AM L ratio was evident after CR. The study of the method and first results obt ained in patient samples support that quantitative real-time PCR with hybri dization probes is a new reliable and sensitive method to monitor minimal r esidual disease in AML patients. Moreover, the fluorescent probes with the LightCycler technology offer the advantage of a rapid detection.