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.

  • Original Manuscript
  • Published:

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

Leukemia volume 17, pages 1051–1057 (2003)Cite this article

Abstract

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 105 polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2

Similar content being viewed by others

References

  1. Vescio RA, Cao J, Hong CH, Lee JC, Wu CH, Der DM et al. Myeloma Ig heavy chain V region sequences reveal prior antigenic selection and marked somatic mutation but no intraclonal diversity. J Immunol 1995; 155: 2487–2497.

    CAS  Google Scholar 

  2. Kosmas C, Stamatopoulos K, Stavroyianni N, Zoi K, Belessi C, Viniou N et al. Origin and diversification of the clonogenic cell in multiple myeloma: lessons from the immunoglobulin repertoire. Leukemia 2000; 14: 1718–1726.

    Article  CAS  Google Scholar 

  3. Pilarski LM, Masellis S, Szczepek A, Mant MJ, Belch AR . Circulating clonotypic B cells in the biology of multiple myeloma: speculations on the origin of myeloma. Leukemia Lymphoma 1996; 22: 375–383.

    Article  CAS  Google Scholar 

  4. Bakkus MH, Heirman C, van Riet I, van Camp B, Thielemans K . Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 1992; 80: 2326–2335.

    CAS  Google Scholar 

  5. Fermand JP, Ravaud P, Chevret S, Divine M, Leblond V, Belanger C et al. High-dose therapy and autologous peripheral blood stem cell transplantation in multiple myeloma: up-front or rescue treatment? Results of a multicenter sequential randomized clinical trial. Blood 1998; 92: 3131–3136.

    CAS  PubMed  Google Scholar 

  6. Dreyfus F, Ribrag V, Leblond V, Ravaud P, Melle J, Quarre MC et al. Detection of malignant B cells in peripheral blood stem cell collections after chemotherapy in patients with multiple myeloma. Bone Marrow Transplant 1995; 15: 707–711.

    CAS  PubMed  Google Scholar 

  7. Corradini P, Voena C, Astolfi M, Ladetto M, Tarella C, Boccadoro M et al. High-dose sequential chemoradiotherapy in multiple myeloma: residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting. Blood 1995; 85: 1596–1602.

    CAS  PubMed  Google Scholar 

  8. Barlogie B, Jagannath S, Desikan KR, Mattox S, Vesole D, Siegel D et al. Total therapy with tandem transplants for newly diagnosed multiple myeloma. Blood 1999; 93: 55–65.

    CAS  PubMed  Google Scholar 

  9. Corradini P, Voena C, Tarella C, Astolfi M, Ladetto M, Palumbo A et al. Molecular and clinical remissions in multiple myeloma: role of autologous and allogeneic transplantation of hematopoietic cells. J Clin Oncol 1999; 17: 208–215.

    Article  CAS  Google Scholar 

  10. Lopez-Perez R, Garcia-Sanz R, Gonzalez D, Balanzategui A, Chillon MC, Alaejos I et al. The detection of contaminating clonal cells in apheresis products is related to response and outcome in multiple myeloma undergoing autologous peripheral blood stem cell transplantation. Leukemia 2000; 14: 1493–1499.

    Article  CAS  Google Scholar 

  11. Bergsagel PL, Smith AM, Szczepek A, Mant MJ, Belch AR, Pilarski LM . In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain. Blood 1995; 85: 436–447.

    CAS  PubMed  Google Scholar 

  12. Vescio RA, Han EJ, Schiller GJ, Lee JC, Wu CH, Cao J et al. Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leukapheresis autografts. Bone Marrow Transplant 1996; 18: 103–110.

    CAS  PubMed  Google Scholar 

  13. Billadeau D, Quam L, Thomas W, Kay N, Greipp P, Kyle R et al. Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients. Blood 1992; 80: 1818–1824.

    CAS  PubMed  Google Scholar 

  14. Ladetto M, Donovan JW, Harig S, Trojan A, Poor C, Schlossnan R et al. Real-Time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma. Biol Blood Marrow Transplant 2000; 6: 241–253.

    Article  CAS  Google Scholar 

  15. Gerard CJ, Olsson K, Ramanathan R, Reading C, Hanania EG . Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards. Cancer Res 1998; 58: 3957–3964.

    CAS  PubMed  Google Scholar 

  16. Rasmussen T, Poulsen TS, Honore L, Johnsen HE . Quantitation of minimal residual disease in multiple myeloma using an allele-specific real-time PCR assay. Exp Hematol 2000; 28: 1039–1045.

    Article  CAS  Google Scholar 

  17. Bruggemann M, Droese J, Bolz I, Luth P, Pott C, von Neuhoff N et al. Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR. Leukemia 2000; 14: 1419–1425.

    Article  CAS  Google Scholar 

  18. Pongers-Willemse MJ, Verhagen OJ, Tibbe GJ, Wijkhuijs AJ, de Haas V, Roovers E et al. Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia 1998; 12: 2006–2014.

    Article  CAS  Google Scholar 

  19. Garcia-Sanz R, Lopez-Perez R, Langerak AW, Gonzalez D, Chillon MC, Balanzategui A et al. Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma. Haematologica 1999; 84: 328–335.

    CAS  PubMed  Google Scholar 

  20. Verhagen OJ, Willemse MJ, Breunis WB, Wijkhuijs AJ, Jacobs DC, Joosten SA et al. Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia. Leukemia 2000; 14: 1426–1435.

    Article  CAS  Google Scholar 

  21. Gonzalez M, Gonzalez D, Lopez-Perez R, Garcia-Sanz R, Chillon MC, Balanzategui A et al. Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies. Haematologica 1999; 84: 779–784.

    CAS  PubMed  Google Scholar 

  22. Cook GP, Tomlinson IM . The human immunoglobulin VH repertoire. Immunol Today 1995; 16: 237–242.

    Article  CAS  Google Scholar 

  23. Lefranc MP . IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 2001; 29: 207–209.

    Article  CAS  Google Scholar 

  24. Schutz E, von A . Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches. Biotechniques 1999; 27: 1218–1222, 1224.

    Article  CAS  Google Scholar 

  25. Schiller G, Vescio R, Freytes C, Spitzer G, Sahebi F, Lee M et al. Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 1995; 86: 390–397.

    CAS  PubMed  Google Scholar 

  26. Henry JM, Sykes PJ, Brisco MJ, To LB, Juttner CA, Morley AA . Comparison of myeloma cell contamination of bone marrow and peripheral blood stem cell harvests. Br J Haematol 1996; 92: 614–619.

    Article  CAS  Google Scholar 

  27. Drunat S, Olivi M, Brunie G, Grandchamp B, Vilmer E, Bieche I et al. Quantification of TEL-AML1 transcript for minimal residual disease assessment in childhood acute lymphoblastic leukaemia. Br J Haematol 2001; 114: 281–289.

    Article  CAS  Google Scholar 

  28. Marcucci G, Caligiuri MA, Dohner H, Archer KJ, Schlenk RF, Dohner K et al. Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia. Leukemia 2001; 15: 1072–1080.

    Article  CAS  Google Scholar 

  29. Cassinat B, Zassadowski F, Balitrand N, Barbey C, Rain JD, Fenaux P et al. Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t(15;17) translocation using real-time RT-PCR. Leukemia 2000; 14: 324–328.

    Article  CAS  Google Scholar 

  30. Wattjes MP, Krauter J, Nagel S, Heidenreich O, Ganser A, Heil G . Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia. Leukemia 2000; 14: 329–335.

    Article  CAS  Google Scholar 

  31. Pfitzner T, Engert A, Wittor H, Schinkothe T, Oberhauser F, Schulz H et al. A real-time PCR assay for the quantification of residual malignant cells in B cell chronic lymphatic leukemia. Leukemia 2000; 14: 754–766.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was partially supported by the BIOMED-2 Concerted Action (BMH4-CT98-3936). David González is supported by the ‘Instituto de Salud Carlos III’ (BISCIII) Grant 99/4230.

Author information

Authors and Affiliations

  1. Department of Hematology, University Hospital of Salamanca, Salamanca, Spain

    D González, M González, M E Alonso, A Balanzategui, M C Chillón, M Silva, R García-Sanz & J F San Miguel

  2. Centro de Investigación del Cáncer (CIC), University of Salamanca, Salamanca, Spain

    R López-Pérez & J F San Miguel

Authors
  1. D González
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M González
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M E Alonso
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R López-Pérez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A Balanzategui
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M C Chillón
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. M Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R García-Sanz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J F San Miguel
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

González, D., González, M., Alonso, M. et al. Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR. Leukemia 17, 1051–1057 (2003). https://doi.org/10.1038/sj.leu.2402937

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2402937

Keywords

This article is cited by

Search

Advanced search

Quick links