Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients

Leukemia. 2002 Dec;16(12):2447-53. doi: 10.1038/sj.leu.2402730.

Abstract

Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the majority of patients but were positive by nested, competitive RT-PCR in 44.6% (n = 29) of samples analyzed (n = 65). These findings indicate that real-time RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy, but we suggest that nested, competitive RT-PCR be used to determine BCR-ABL/ABL transcript ratios at low transcript values or especially when real-time analyses are negative.

Publication types

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

MeSH terms

  • Fusion Proteins, bcr-abl / genetics*
  • Humans
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / diagnosis
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / genetics*
  • Neoplasm, Residual / diagnosis
  • Neoplasm, Residual / genetics
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • RNA, Neoplasm / analysis*
  • Sensitivity and Specificity
  • Tumor Cells, Cultured

Substances

  • RNA, Neoplasm
  • Fusion Proteins, bcr-abl