Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a'real time' quantitative RT-PCR assay

Quantitative competitive RT-PCR techniques have been developed to detect BC R-ABL fusion transcripts in CML but they are hardly reproducible. In this w ork, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rap id quantification of a target sequence during the extension phase of PCR am plifications. A fluorogenic probe labeled with both a reporter dye at the 5 ' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Tag DNA po lymerase cleaves the probe and releases the reporter dye, resulting in an i ncrease in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA st andards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, th e sensitivity of a serial dilution of total RNA from a positive cell line ( K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cy togenetic CR, including 11 allografted patients, two autografted patients a nd two patients treated by IFN were studied sequentially by this new real t ime quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ ABL ratio in all samples. The two patients treated by IFN showed a progress ive decrease in the ratio. In the II allografted patients, four were sequen tially studied 2 years or more after allo-BMT, and all ratios were below 10 -4. The four patients remained in clinical and cytogenetic CR. The seven ot her allografted patients were studied immediately after the procedure. Thre e of them showed a progressive decrease in the BCR-ABL/ABL ratio which reac hed 10(-4) 7 months after allo-BMT. The three patients remained in hematolo gic and cytogenetic CR. The remaining four allografted patients had progres sive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR i n the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round n ested PCR. However, evolution changes in the results of real-time quantitat ive RT-PCR often preceded those of the conventional technique: a decrease o f the BCR-ABL/ABL ratio preceded progression from first round to second rou nd positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinic al and cytogenetic evolution than conventional qualitative techniques and m ay help in making early therapeutic decisions in CML, especially after mole cular relapse.