Quantitative detection of AML1-ETO rearrangement by real-time RT-PCR using fluorescently labeled probes.
The persistence of the AML1-ETO rearrangement performed by reverse transcription polymerase chain reaction (RT-PCR) has been reported in acute myeloid leukemia (AML) patients in long-term complete remission (CR). This persistence, which is not associated with hematological relapse, limits the clinical use of qualitative RT-PCR. Here, we present a new quantitative real-time PCR method to detect AML1-ETO rearrangement using fluorescently labeled probes. Quantitative detection of AML1-ETO was performed in capillary tubes using two fluorescently labeled probes in the LightCycler equipment. The reliability of the method was checked in twenty-two bone marrow samples and one apheresis sample from eight patients with t(8;21) collected at diagnosis and during follow-up assessment. The regression coefficients obtained for standard curves of AML1-ETO and AML were all greater than 0.98. The sensitivity 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-ETO, and 7% for AML. The inter-assay coefficient of variation was 19% for AML1-ETO and 12% for AML. A log reduction from two to four in the AML1-ETO/AML ratio was evident after CR. The study of the method and first results obtained in patient samples support that quantitative real-time PCR with hybridization probes is a new reliable and sensitive method to monitor minimal residual disease in AML patients. Moreover, the fluorescent probes with the Light-Cycler technology offer the advantage of a rapid detection.
Barragán
E
E
Department of Clinical Pathology, Hospital Universitario La Fe Valencia, Spain.
Bolufer
P
P
Moreno
I
I
Martín
G
G
Nomdedeu
J
J
Brunet
S
S
Fernández
P
P
Rivas
C
C
Sanz
M A
MA
D000208:Acute Disease; D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D050676:Core Binding Factor Alpha 2 Subunit; D015342:DNA Probes; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D007951:Leukemia, Myeloid / Q000175:diagnosis / Q000235:genetics; D008297:Male; D008875:Middle Aged; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D012334:RNA, Neoplasm / Q000032:analysis*; D000075142:RUNX1 Translocation Partner 1 Protein; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics*; D014178:Translocation, Genetic
C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; D015342:DNA Probes; D005456:Fluorescent Dyes; D015514:Oncogene Proteins, Fusion; D012334:RNA, Neoplasm; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors