Evaluation of single tube DzyNA-RTPCR as a tool for real-time quantification of PML/RARα fusion transcripts in Acute Promyelocytic Leukaemia.

TL Applegate BSca, HJ Iland MBBS b, AV Todd PhD a

a Johnson & Johnson Research Pty Limited, Sydney, NSW Australia b Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, Sydney, NSW Australia.

AIM To develop DzyNA-RTPCR to allow real-time fluorescent detection and quantification of fusion transcripts and evaluate the clinical feasibility of this assay to monitor minimal residual disease in Acute Promyelocytic Leukaemia (APL). METHODS Single tube DzyNA-RTPCR contains all the reagents required for amplification, detection and quantification of mRNA. DzyNA-RTPCR amplicons contain target sequences linked to catalytic deoxyribozymes. Amplicons cleave generic reporter substrates during PCR and resultant increases in fluorescence are monitored in real time (Clin Chem 2000: 46, 625-30). RESULTS Parallel DzyNA-RTPCR assays were developed to quantify the disease fusion transcript, PML/RARα and a low abundance control transcript, normal BCR. Total RNA from the human NB4 cell line was used to generate calibration curves and estimate both transcripts in total RNA extracted from bone marrow of patients with APL. DzyNA-RTPCR detected low numbers of transcripts and allowed accurate and reproducible quantification of the specific transcripts over a broad linear range. PML/RARα mRNA was quantified in ten patients at diagnosis and in one patient over a seven-year period. Monitoring of transcript levels effectively reflected the disease course over time. The high levels observed at diagnosis fell to undetectable levels during remission following induction treatment. Levels subsequently increased heralding a molecular relapse that preceded a hematological relapse by several months. This patient achieved a second remission following salvage therapy and continues to be monitored. CONCLUSIONS DzyNA-RTPCR is suitable for use in clinical practice as a tool for diagnosis and for subsequent monitoring of minimal residual disease and detection of molecular relapse in APL.

KEY WORDS: generic substrate, minimal residual disease, clinical feasibility, molecular relapse, DzyNA PCR.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Chromosomal Aberrations.