Skip to main content
SpringerLink
Log in
Book cover

Lymphoma pp 175–200Cite as

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 971))

Abstract

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell Non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is the gold-standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). RQ-PCR analysis of the junctional regions of the rearranged immunoglobulin heavy-chain gene (IgH) serves as the most broadly applicable MRD target in B-NHL (∼80%). Chromosomal translocations as t(14;18) translocation in FL and t(11;14) translocation in MCL can be used in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry as well as RQ-PCR are equally suitable for MRD assessment as long as a sensitivity of ≤10−4 shall be achieved.

MRD diagnostics targeting the IgH gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. Furthermore, somatic mutations of the IgH region occurring during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IgH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10−4). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IgH gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Brown JR, Feng Y, Gribben JG, Neuberg D, Fisher DC, Mauch P et al (2007) Long-term survival after autologous bone marrow transplantation for follicular lymphoma in first remission. Biol Blood Marrow Transplant 13:1057–1065

    Article  PubMed  Google Scholar 

  2. Goff L, Summers K, Iqbal S, Kuhlmann J, Kunz M, Louton T et al (2009) Quantitative PCR analysis for Bcl-2/IgH in a phase III study of Yttrium-90 Ibritumomab Tiuxetan as consolidation of first remission in patients with follicular lymphoma. J Clin Oncol 27:6094–6100

    Article  PubMed  CAS  Google Scholar 

  3. Hardingham JE, Kotasek D, Sage RE, Gooley LT, Mi JX, Dobrovic A et al (1995) Significance of molecular marker-positive cells after autologous peripheral-blood stem-cell transplantation for non-Hodgkin’s lymphoma. J Clin Oncol 13:1073–1079

    PubMed  CAS  Google Scholar 

  4. Hirt C, Schuler F, Dolken G (2003) Minimal residual disease (MRD) in follicular lymphoma in the era of immunotherapy with rituximab. Semin Cancer Biol 13:223–231

    Article  PubMed  CAS  Google Scholar 

  5. Ladetto M, Magni M, Pagliano G, De Marco F, Drandi D, Ricca I et al (2006) Rituximab induces effective clearance of minimal residual disease in molecular relapses of mantle cell lymphoma. Biol Blood Marrow Transplant 12:1270–1276

    Article  PubMed  CAS  Google Scholar 

  6. Moos M, Schulz R, Martin S, Benner A, Haas R (1998) The remission status before and the PCR status after high-dose therapy with peripheral blood stem cell support are prognostic factors for relapse-free survival in patients with follicular non-Hodgkin’s lymphoma. Leukemia 12:1971–1976

    Article  PubMed  CAS  Google Scholar 

  7. Moreton P, Kennedy B, Lucas G, Leach M, Rassam SMB, Haynes A et al (2005) Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after Alemtuzumab therapy Is associated with prolonged survival. J Clin Oncol 23:2971–2979

    Article  PubMed  CAS  Google Scholar 

  8. Pott C, Hoster E, Beldjord K, Macintyre E, Böttcher S, Asnafi V et al (2010) R-CHOP/R-DHAP prior to autologous stem cell transplantation induces higher rates of molecular remission than R-CHOP in younger patients with MCL: results of the MCL younger intergroup trial of the European MCL Network. Blood 116: abstract 528

    Google Scholar 

  9. Pott C, Hoster E, Delfau-Larue MH, Beldjord K, Bottcher S, Asnafi V et al (2010) Molecular remission is an independent predictor of clinical outcome in patients with mantle cell lymphoma after combined immunochemotherapy: a European MCL intergroup study. Blood 115:3215–3223

    Article  PubMed  CAS  Google Scholar 

  10. Pott C, Schrader C, Gesk S, Harder L, Tiemann M, Raff T et al (2006) Quantitative assessment of molecular remission after high-dose therapy with autologous stem cell transplantation predicts long-term remission in mantle cell lymphoma. Blood 107:2271–2278

    Article  PubMed  CAS  Google Scholar 

  11. Provan D, Bartlett-Pandite L, Zwicky C, Neuberg D, Maddocks A, Corradini P et al (1996) Eradication of polymerase chain reaction-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation. Blood 88:2228–2235

    PubMed  CAS  Google Scholar 

  12. Geisler CH, Kolstad A, Laurell A, Andersen NS, Pedersen LB, Jerkeman M et al (2008) Long-term progression-free survival of mantle cell lymphoma after intensive front-line immunochemotherapy with in vivo-purged stem cell rescue: a nonrandomized phase 2 multicenter study by the Nordic Lymphoma Group. Blood 112:2687–2693

    Article  PubMed  CAS  Google Scholar 

  13. Ladetto M, De Marco F, Benedetti F, Vitolo U, Patti C, Rambaldi A et al (2008) Prospective, multicenter randomized GITMO/IIL trial comparing intensive (R-HDS) versus conventional (CHOP-R) chemoimmunotherapy in high-risk follicular lymphoma at diagnosis: the superior disease control of R-HDS does not translate into an overall survival advantage. Blood 111:4004–4013

    Article  PubMed  CAS  Google Scholar 

  14. Rambaldi A, Carlotti E, Oldani E, Della SI, Baccarani M, Cortelazzo S et al (2005) Quantitative PCR of bone marrow BCL2/IgH positive cells at diagnosis predicts treatment response and long-term outcome in Follicular non Hodgkin’s Lymphoma. Blood 105:3428–3433

    Article  PubMed  CAS  Google Scholar 

  15. Andersen NS, Donovan JW, Zuckerman A, Pedersen L, Geisler C, Gribben JG (2002) Real-time polymerase chain reaction estimation of bone marrow tumor burden using clonal immunoglobulin heavy chain gene and bcl-1/JH rearrangements in mantle cell lymphoma. Exp Hematol 30:703–710

    Article  PubMed  CAS  Google Scholar 

  16. Andersen NS, Pedersen LB, Laurell A, Elonen E, Kolstad A, Boesen AM et al (2009) Pre-emptive treatment with rituximab of molecular relapse after autologous stem cell transplantation in mantle cell lymphoma. J Clin Oncol 27:4365–4370

    Article  PubMed  CAS  Google Scholar 

  17. Rambaldi A, Lazzari M, Manzoni C, Carlotti E, Arcaini L, Baccarani M et al (2002) Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma. Blood 99:856–862

    Article  PubMed  CAS  Google Scholar 

  18. Evans PAS, Pott C, Groenen PJTA, Salles G, Davi F, Berger F et al (2006) Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 21:207–214

    Article  PubMed  Google Scholar 

  19. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL et al (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17:2257–2317

    Article  PubMed  Google Scholar 

  20. Kneba M, Bergholz M, Bolz I, Hulpke M, Batge R, Schauer A et al (1990) Heterogeneity of immunoglobulin gene rearrangements in B-cell lymphomas. Int J Cancer 45:609–613

    Article  PubMed  CAS  Google Scholar 

  21. Kneba M, Eick S, Herbst H, Willigeroth S, Pott C, Bolz I et al (1991) Frequency and structure of t(14;18) major breakpoint regions in non-Hodgkin’s lymphomas typed according to the Kiel classification: analysis by direct DNA sequencing. Cancer Res 51:3243–3250

    PubMed  CAS  Google Scholar 

  22. Pott C, Tiemann M, Linke B, Ott MM, von Hofen M, Bolz I et al (1998) Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas. Leukemia 12:1630–1637

    Article  PubMed  CAS  Google Scholar 

  23. van der Velden V, van Dongen J (2009) MRD detection in acute lymphoblastic leukemia patients using Ig/TCR gene rearrangements as targets for real-time quantitative PCR. In: Walker JM (ed) Lymphoma: methods and protocols. Humana Press, p. 115–150

    Google Scholar 

  24. Olsson K, Gerard CJ, Zehnder J, Jones C, Ramanathan R, Reading C et al (1999) Real-time t(11;14) and t(14;18) PCR assays provide sensitive and quantitative assessments of minimal residual disease (MRD). Leukemia 13:1833–1842

    Article  PubMed  CAS  Google Scholar 

  25. Pott C, Schrader C, Trautmann H, Irmer S, Desgranges C, Ritgen M et al (2003) VH mutational status and VH gene usage define molecular heterogeneity in mantle cell lymphoma. Onkologie 26(suppl 5):162

    Google Scholar 

  26. van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ (2003) Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. Leukemia 17:1013–1034

    Article  PubMed  Google Scholar 

  27. van der Velden VHJ, Cazzaniga G, Schrauder A, Hancock J, Bader P, Panzer-Grumayer ER et al (2007) Analysis of minimal residual disease by Ig//TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 21:604–611

    PubMed  Google Scholar 

  28. Payne K, Wright P, Grant JW, Huang Y, Hamoudi R, Bacon CM et al (2011) BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma. Br J Haematol 155:84–92

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  30. Donovan JW, Ladetto M, Zou G, Neuberg D, Poor C, Bowers D et al (2000) Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia. Blood 95:2651–2658

    PubMed  CAS  Google Scholar 

  31. Akasaka T, Akasaka H, Yonetani N, Ohno H, Yamabe H, Fukuhara S, Okuma M (1998) Refinement of the BCL2/immunoglobulin heavy chain fusion gene in t(14;18)(q32;q21) by polymerase chain reaction amplification for long targets. Genes Chromosomes Cancer 21:17–29

    Article  PubMed  CAS  Google Scholar 

  32. Buchonnet G, Lenain P, Ruminy P, Lepretre S, Stamatoullas A, Parmentier F et al (2000) Characterisation of BCL2-JH rearrangements in follicular lymphoma: PCR detection of 3′ BCL2 breakpoints and evidence of a new cluster. Leukemia 14:1563–1569

    Article  PubMed  CAS  Google Scholar 

  33. Albinger-Hegyi A, Hochreutener B, Abdou MT, Hegyi I, Dours-Zimmermann MT, Kurrer MO et al (2002) High frequency of t(14;18)-translocation breakpoints outside of major breakpoint and minor cluster regions in follicular lymphomas: improved polymerase chain reaction protocols for their detection. Am J Pathol 160:823–832

    Article  PubMed  CAS  Google Scholar 

  34. van Krieken JHJM, Langerak AW, Macintyre EA, Kneba M, Hodges E, Sanz RG et al (2006) Improved reliability of lymphoma diagnostics via PCR-based clonality testing: [mdash] Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 21:201–206

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge Ariane Stuhr, Karin Olsen, and Petra Chall for their excellent technical support.

Author information

Authors and Affiliations

  1. Second Department of Medicine, University Hospital Schleswig-Holstein, Kiel, Germany

    Christiane Pott & Monika Brüggemann

  2. Second Department of Medicine, University Hospital of Schleswig-Holstein, Kiel, Germany

    Matthias Ritgen & Michael Kneba

  3. Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

    Vincent H. J. van der Velden & Jacques J. M. van Dongen

Authors
  1. Christiane Pott
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Monika Brüggemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Ritgen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vincent H. J. van der Velden
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jacques J. M. van Dongen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Kneba
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christiane Pott .

Editor information

Editors and Affiliations

  1. Medical School, Institute of Cell Biology (Cancer Resear, University of Duisburg-Essen, Virchowstr. 173, Essen, 45122, Germany

    Ralf Küppers

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Pott, C., Brüggemann, M., Ritgen, M., van der Velden, V.H.J., van Dongen, J.J.M., Kneba, M. (2013). MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 971. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-269-8_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-269-8_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-268-1

  • Online ISBN: 978-1-62703-269-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation