Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Biotechnical Methods Section BTS
  • Published:

Biotechnical Methods Section (BTS)

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

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 μg 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 μg 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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1

Similar content being viewed by others

References

  1. Kloetzer W, Kurzrock R, Smith L, Talpaz M, Spiller M, Gutterman J, Arlinghaus R . The human cellular abl gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine kinase activity Virology 1985 140: 230–238

    Article  CAS  Google Scholar 

  2. Davis RL, Konopka JB, Witte ON . Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties Mol Cell Biol 1985 5: 204–213

    Article  CAS  Google Scholar 

  3. Rowley JD . A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining Nature 1973 243: 290–293

    Article  CAS  Google Scholar 

  4. Shtivelman E, Lifshitz B, Gale RP, Canaani E . Fused transcript of abl and bcr genes in chronic myelogenous leukaemia Nature 1985 315: 550–553

    Article  CAS  Google Scholar 

  5. Tkachuk DC, Westbrook CA, Andreeff M, Donlon DA, Cleary ML, Surynanarayan K, Home R, Redner A, Gray J, Pinkel D . Detection of bcr-abl fusion in chronic myelogenous leukemia by in situ hybridization Science 1990 250: 559–562

    Article  CAS  Google Scholar 

  6. Shtalrid M, Talpaz M, Kurzrock R, Kantarjian H, Trujillo J, Gutterman J, Yoffe G, Blick M . Analysis of breakpoints within the bcr gene and their correlation with the clinical course of Philadelphia-positive chronic myelogenous leukemia Blood 1988 72: 485–490

    Article  CAS  Google Scholar 

  7. Guo JQ, Wang JY, Arlinghaus RB . Detection of BCR-ABL proteins in blood cells of benign phase chronic myelogenous leukemia patients Cancer Res 1991 51: 3048–3051

    CAS  PubMed  Google Scholar 

  8. Guo JQ, Lian JY, Xian YM, Lee MS, Deisseroth AB, Stass SA, Champlin RE, Talpaz M, Wang JY, Arlinghaus RB . BCR-ABL protein expression in peripheral blood cells of chronic myelogenous leukemia patients undergoing therapy Blood 1994 15: 3629–3637

    Article  Google Scholar 

  9. Cross NC, Feng L, Bungey J, Goldman JM . Minimal residual disease after bone marrow transplant for chronic myeloid leukemia detected by the polymerase chain reaction Leuk Lymphoma 1993 11: 39–43

    Article  Google Scholar 

  10. Cross NC, Hughes TP, Feng L, O'Shea P, Bungey J, Marks DI, Ferrant A, Martiat P, Goldman JM . Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: correlations with acute graft-versus-host disease and relapse Br J Haematol 1993 1: 67–74

    Article  Google Scholar 

  11. Hochhaus A, Weisser A, La Rosee P, Emig M, Muller MC, Saussele S, Reiter A, Kuhn C, Berger U, Hehlmann R, Cross NC . Detection and quantification of residual disease in chronic myelogenous leukemia Leukemia 2000 6: 998–1005

    Article  Google Scholar 

  12. Cross NC, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM . Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation Blood 1993 82: 1929–1936

    Article  CAS  Google Scholar 

  13. Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A, Berger U, Hehlmann R, Cross NC, Hochhaus A . Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR Leukemia 1999 11: 1825–1832

    Article  Google Scholar 

  14. Eder M, Battmer K, Kafert S, Stucki A, Ganser A, Hertenstein B . Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation Leukemia 1999 13: 1383–1389

    Article  CAS  Google Scholar 

  15. Branford S, Hughes TP, Rudzki Z . Monitoring chronic myeloid leukemia therapy by real-time quantitative PCR in blood is reliable alternative to bone marrow cytogenetics Br J Haematol 1999 107: 587–599

    Article  CAS  Google Scholar 

  16. Lozzio CB, Lozzio BB . Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome Blood 1975 45: 321–334

    Article  CAS  Google Scholar 

  17. Andersson BS, Collins VP, Kurzrock R, Larkin DW, Childs C, Ost A, Cork A, Trujillo JM, Freireich EJ, Siciliano MJ, Deisseroth AB . KBM-7, a human myeloid leukemia cell line with double Philadelphia chromosomes lacking normal c-ABL and BCR transcripts Leukemia 1995 9: 2100–2108

    CAS  PubMed  Google Scholar 

  18. Koeffler HP, Lowe L, Golde DW . Amygdalin (Laetrile): effect on clonogenic cells from human myeloid leukemia cell lines and normal human marrow Cancer Treat Rep 1980 1: 105–109

    Google Scholar 

  19. Smith RG, Dev VG, Shannon WA Jr . Characterization for a novel human pre-B leukemia cell line J Immunol 1981 126: 596–602

    CAS  PubMed  Google Scholar 

  20. Cross NC, Melo JV, Feng L, Goldman JM . An optimized multiplex polymerase chain reaction (PCR) for detection of BCR-ABL fusion mRNAs in haematological disorders Leukemia 1994 1: 186–189

    Google Scholar 

  21. Hochhaus A, Lin F, Reiter A, Skladny H, Hehlmann R, Goldman JM, Cross NC . Monitoring the efficiency of interferon-alpha therapy in chronic myelogenous leukemia (CML) patients by competitive polymerase chain reaction Leukemia 1997 11(Suppl. 3): 541–544

    Google Scholar 

  22. Seong D, Kantarjian H, Ro J, Siciliano M . Hypermetaphase FISH for quantitative monitoring of Ph-positive cells in patients With CML during treatment Blood 1995 86: 2343–2349

    Article  CAS  Google Scholar 

  23. Faderl S, Talpaz M, Kantarjian HM, Estrov Z . Should polymerase chain reaction analysis to detect minimal residual disease in patients with chronic myelogenous leukemia be used in clinical decision making Blood 1999 9: 2755–2759

    Article  Google Scholar 

  24. Olavarria E, Kanfer E, Szydlo R, Kaeda J, Rezvani K, Cwynarski K, Pocock C, Dazzi F, Craddock C, Apperley JF, Cross NC, Goldman JM . Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia Blood 2001 97: 1560–1565

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Dr Goldman for the competitor plasmids, and Dr Feng Lin for advice and suggestions. In addition, we would like to thank Dr Ke Si for help in statistical analysis and we acknowledge expert technical assistance of Qing Wang and Jialing Xu. This research was supported by grants from NIH (CA49639) and the Hendricks Foundation.

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, J., Lin, H., Kantarjian, H. et al. Comparison of competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients. Leukemia 16, 2447–2453 (2002). https://doi.org/10.1038/sj.leu.2402730

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2402730

Keywords

This article is cited by

Search

Quick links