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

Abstract

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, 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 rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5Ј end and a quencherdye at the 3Ј end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase 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 standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the 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 cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time 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 progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

“…To obtain the standard curve, laboratories used either cell line cDNA 37,39,128,190 or plasmid DNA. 38,42,125,191 So far, few authors used the DDCt method 49,192 and, to our knowledge, a standard curve of diagnostic cDNA has not been used so far. In our EAC network, we discussed four possibilities: (1) cell line RNA dilutions, (2) percentage of positive cell number relative to the diagnostic sample, (3) copy number ratios and (4) the DDCt method.…”

“…35,36 There have been numerous manuscripts from individual laboratories demonstrating the reliability of this technology and its potential clinical value for MRD studies using FG transcripts as PCR targets such as BCR-ABL in CML [37][38][39] or ALL, 40 PML-RARA, 41 AML1-ETO [42][43][44] and CBFB-MYH11 45,46 in AML and TEL-AML1 [47][48][49] in ALL patients. However, standardized RQ-PCR procedures are warranted in order to apply this innovative technology for large-scale MRD studies within multicenter therapeutic trials.…”

“…Since the advent of real-time PCR, several groups have published reports that describe the feasibility of monitoring CML patients with this technique. [37][38][39][125][126][127] However, most RQ-PCR studies included too few patients or patients from different therapeutic protocols that together with methodological differences make it difficult to evaluate the clinical impact and to define general guidelines to monitor CML patients with RQ-PCR. The availability of a standardized protocol for RQ-PCR will facilitate data comparison among different centers, making it possible to define a threshold where a patient is likely to relapse and ultimately to assess the impact of an early therapeutic intervention based on the kinetics of BCR-ABL FG transcripts.…”

True

“…5,6 In this study we have developed a related system using fluorescent hybridization probes in the LightCycler. 8,30 One advantage of this system is the rapid cycling time (Ͻ40 s/cycle) due to amplification being performed in glass capillaries and being driven by forced air heating.…”

“…A procedure for quantification of BCR-ABL mRNA using the TaqMan system has been described. 5,6 Here we have established an alternative real-time RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the new LightCycler technology, 7,8 which combines rapid thermocycling with online fluorescence detection of PCR product formation as it occurs. Fluorescence monitoring of PCR amplification is based on the concept of fluorescence resonance energy transfer (FRET) between two adjacent dyes.…”