Detection of bcr-abl transcript in chronic myelogenous leukemia patients by reverse-transcription-polymerase chain reaction and capillary electrophoresis

Abstract

BACKGROUND AND OBJECTIVE: Capillary electrophoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis). DESIGN AND METHODS: The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 microgram of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per microgram of total RNA examined). RESULTS: CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two types of bcr-abl junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (SD) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/microgram total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value 128,859 vs. 331,722 in patients with 0% blast cells and > 1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score < 0.8 and > 1.2, respectively; p = 0.003). INTERPRETATION AND CONCLUSIONS: Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.