Rapid quantitative detection of TEL-AML1 fusion transcripts in pediatric acute lymphoblastic leukemia by real-time reverse transcription polymerase chain reaction using fluorescently labeled probes
Bolufer, P.; Barragán, E.; Verdeguer, A.; Cervera, Jé.; Fernández, Jé.María.; Moreno, I.; Lerma, E.; Esquembre, C.; Tasso, Mía.; Fuster, V.; Bermúdez, M.; Sanz, M.A.
Haematologica 87(1): 23-32
2002
ISSN/ISBN: 0390-6078 PMID: 11801462 Document Number: 546112
Background and Objectives: We report a new real-time reverse transcription polymerase chain reaction (RT-PCR) method for quantification of TEL-AML1 transcripts. The method is based on hybridization probe (HybProbe) chemistry applied in LightCycler equipment. The study group comprised 44 successive cases of pediatric acute lymphoblastic leukemia (P-ALL). Design and Methods: The quantitative estimation of TEL-AML1 transcripts was performed in 10 P-ALL TEL-AML1-positive patients. The PCR was performed in capillary tubes in 10 muL final volumes using two sets of primers: M1, which detects the long (L-form) and short (S-form) transcripts, and M2, specific for the L-form. The fluorescently labeled HybProbes (TEL 3FL and TEL 5LC) hybridize to the TEL region. TEL-AML1 expression was normalized relative to the levels of AML1 transcripts. Standard curves were prepared using serial dilutions of plasmids with TEL-AML1 or AML1 inserts. Results: The sensitivity attained allowed the detection of TEL-AML1 transcripts at a 10-4 dilution of a cDNA sample from a patient at diagnosis. The within-assay coefficient of variation (CV) for TEL-AML1 was 7.0% and the between-assay CV was 13%. Levels of TEL-AML1 transcript and the TEL-AML1/AML1 ratio decreased by more than four log units (p<0.001) during or after the course of initial treatment. Most of the patients who achieved complete remission after 5-6 months of initial treatment were TEL-AML1 negative, although some positive samples with negligible amounts of TEL-AML1 transcripts were still detected. Interpretation and Conclusions: This method has the sensitivity and reliability required to monitor the presence of minimal residual disease, and could be a powerful tool in monitoring the efficacy of the response to chemotherapy.