title: Design and feasibility of a novel, rapid, and simple fluorescence 26-plex rt-PCR assay for simultaneous detection of 24 fusion transcripts in adult acute myeloid leukemia.
abstract: Identification of chromosomal abnormalities is mandatory for classification of acute myeloid leukemia (AML), and the abnormalities have to be determined quickly, to allow patient enrollment in multicenter protocols and/or for selecting therapeutic strategies. Rapid AML molecular diagnosis is often difficult to achieve, however, because it is based on numerous different RT-PCR protocols. We developed a new RT-PCR method, one that does not require a nested step, to simultaneously detect all AML fusion transcripts from six major recurrent translocations found in adults: t(15;17)(q22;q12), inv(16)(p13.1q22) [t(16;16)(p13.1;q22)], t(8;21)(q22;q22), t(6;9)(p23;q34), t(9;22)(q34;q11), and t(10;11)(p13;q14). Specific primers for RT-PCR detection of the 24 fusion transcripts, along with two transcripts for controls, were designed for this 26-plex RT-PCR. Each PCR product had a different size and was separated by capillary electrophoresis. We also designed a multiplex positive control with 24 chimeric RNAs, corresponding to all chimeric RNAs tested. Compared with classical molecular biology protocols and cytogenetic analyses used as reference standards, results of the 26-plex RT-PCR method were concordant in all 204 (100%) cases of adult AML tested. Results were obtained in less than 24 hours. Because of the multiplex positive control, interpretation of the peaks was very easy, without any ambiguity. The tumor cell detection threshold was 1.5%.
authors: Laboratory of Hematology and UMR CNR 7276, CHU Dupuytren and Faculty of Medicine, University of Limoges, Limoges, France.
authors: Marie-Pierre
authors: MP
authors: Laforêt
authors: Pascal
authors: P
authors: Turlure
authors: Eric
authors: E
authors: Lippert
authors: Pascale
authors: P
authors: Cornillet-Lefebvre
authors: Arnaud
authors: A
authors: Pigneux
authors: Rachel
authors: R
authors: Pradeau
authors: Jean
authors: J
authors: Feuillard
authors: Nathalie
authors: N
authors: Gachard
mesh_terms: D000328:Adult; D017931:DNA Primers; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D011786:Quality Control; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014178:Translocation, Genetic
chemical_list: D017931:DNA Primers; D015514:Oncogene Proteins, Fusion