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

Leuk Lymphoma. 2001 Aug;42(4):747-56. doi: 10.3109/10428190109099337.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Disease
  • Adolescent
  • Adult
  • Child
  • Child, Preschool
  • Core Binding Factor Alpha 2 Subunit
  • DNA Probes
  • Female
  • Fluorescent Dyes
  • Humans
  • Leukemia, Myeloid / diagnosis
  • Leukemia, Myeloid / genetics
  • Male
  • Middle Aged
  • Neoplasm, Residual / diagnosis
  • Neoplasm, Residual / genetics
  • Oncogene Proteins, Fusion / genetics*
  • RNA, Neoplasm / analysis*
  • RUNX1 Translocation Partner 1 Protein
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / standards*
  • Sensitivity and Specificity
  • Transcription Factors / genetics*
  • Translocation, Genetic

Substances

  • AML1-ETO fusion protein, human
  • Core Binding Factor Alpha 2 Subunit
  • DNA Probes
  • Fluorescent Dyes
  • Oncogene Proteins, Fusion
  • RNA, Neoplasm
  • RUNX1 Translocation Partner 1 Protein
  • Transcription Factors