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 Article
  • Published:

Biomed-2

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936

Leukemia volume 21, pages 222–229 (2007)Cite this article

Abstract

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell receptor (TCR) rearrangements can be useful. Here we address the issue of clonality assessment in 106 histologically defined reactive lesions, using the standardized BIOMED-2 Ig/TCR multiplex polymerase chain reaction (PCR) heteroduplex and GeneScan assays. Samples were reviewed nationally, except 10% random cases and cases with clonal results selected for additional international panel review. In total 75% (79/106) only showed polyclonal Ig/TCR targets (type I), whereas another 15% (16/106) represent probably polyclonal cases, with weak Ig/TCR (oligo)clonality in an otherwise polyclonal background (type II). Interestingly, in 10% (11/106) clear monoclonal Ig/TCR products were observed (types III/IV), which prompted further pathological review. Clonal cases included two missed lymphomas in national review and nine cases that could be explained as diagnostically difficult cases or probable lymphomas upon additional review. Our data show that the BIOMED-2 Ig/TCR multiplex PCR assays are very helpful in confirming the polyclonal character in the vast majority of reactive lesions. However, clonality detection in a minority should lead to detailed pathological review, including close interaction between pathologist and molecular biologist.

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. Van Dongen JJM, Langerak AW, Bruggemann M, Evans PAS, Hummel M, Lavender FL et al. 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 2003; 17: 2257–2317.

    Article  CAS  PubMed  Google Scholar 

  2. Sandberg Y, Heule F, Lam K, Lugtenburg PJ, Wolvers-Tettero IL, van Dongen JJ et al. Molecular immunoglobulin/T- cell receptor clonality analysis in cutaneous lymphoproliferations. Experience with the BIOMED-2 standardized polymerase chain reaction protocol. Haematologica 2003; 88: 659–670.

    CAS  PubMed  Google Scholar 

  3. Evans PA, Pott C, Groenen PJ, Salles G, Davi F, Berger F et al. 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 2007, (in press).

  4. Bruggemann M, White HE, Gaulard P, Garcia Sanz R, Gameiro P, Oeschger S et al. Powerful strategy for PCR-based clonality assessment in T-cell malignancies. Report of the BIOMED-2 Concerted Action BHM4 CT98-3936. Leukemia 2007, (in press).

  5. Magro C, Crowson AN, Kovatich A, Burns F . Pityriasis lichenoides: a clonal T-cell lymphoproliferative disorder. Hum Pathol 2002; 33: 788–795.

    Article  PubMed  Google Scholar 

  6. Poenitz N, Dippel E, Klemke CD, Qadoumi M, Goerdt S . Jessner's lymphocytic infiltration of the skin: a CD8+ polyclonal reactive skin condition. Dermatology 2003; 207: 276–284.

    Article  CAS  PubMed  Google Scholar 

  7. Vanni R, Fletcher CD, Sciot R, Dal Cin P, De Wever I, Mandahl N et al. Cytogenetic evidence of clonality in cutaneous benign fibrous histiocytomas: a report of the CHAMP study group. Histopathology 2000; 37: 212–217.

    Article  CAS  PubMed  Google Scholar 

  8. De Vita S, De Re V, Gasparotto D, Ballare M, Pivetta B, Ferraccioli G et al. Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 2000; 43: 94–102.

    Article  CAS  PubMed  Google Scholar 

  9. Hanto DW, Birkenbach M, Frizzera G, Gajl-Peczalska KJ, Simmons RL, Schubach WH . Confirmation of the heterogeneity of posttransplant Epstein–Barr virus-associated B cell proliferations by immunoglobulin gene rearrangement analyses. Transplantation 1989; 47: 458–464.

    Article  CAS  PubMed  Google Scholar 

  10. Langerak AW, van Krieken JH, Wolvers-Tettero IL, Kerkhof E, Mulder AH, Vrints LW et al. The role of molecular analysis of immunoglobulin and T cell receptor gene rearrangements in the diagnosis of lymphoproliferative disorders. J Clin Pathol 2001; 54: 565–567.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Langerak AW, Sandberg Y, van Dongen JJ . Spectrum of T-large granular lymphocyte lymphoproliferations: ranging from expanded activated effector T cells to T-cell leukaemia. Br J Haematol 2003; 123: 561–562.

    Article  PubMed  Google Scholar 

  12. Sinclair E, Black D, Epling CL, Carvidi A, Josefowicz SZ, Bredt BM et al. CMV antigen-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation responses in healthy CMV-seropositive individuals. Viral Immunol 2004; 17: 445–454.

    Article  CAS  PubMed  Google Scholar 

  13. Doisne JM, Urrutia A, Lacabaratz-Porret C, Goujard C, Meyer L, Chaix ML et al. CD8+ T cells specific for EBV, cytomegalovirus, and influenza virus are activated during primary HIV infection. J Immunol 2004; 173: 2410–2418.

    Article  CAS  PubMed  Google Scholar 

  14. van Leeuwen EM, Remmerswaal EB, Vossen MT, Rowshani AT, Wertheim-van Dillen PM, van Lier RA et al. Emergence of a CD4+CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection. J Immunol 2004; 173: 1834–1841.

    Article  CAS  PubMed  Google Scholar 

  15. Gamadia LE, van Leeuwen EM, Remmerswaal EB, Yong SL, Surachno S, Wertheim-van Dillen PM et al. The size and phenotype of virus-specific T cell populations is determined by repetitive antigenic stimulation and environmental cytokines. J Immunol 2004; 172: 6107–6114.

    Article  CAS  PubMed  Google Scholar 

  16. Katchar K, Wahlstrom J, Eklund A, Grunewald J . Highly activated T-cell receptor AV2S3(+) CD4(+) lung T-cell expansions in pulmonary sarcoidosis. Am J Respir Crit Care Med 2001; 163: 1540–1545.

    Article  CAS  PubMed  Google Scholar 

  17. Farzati B, Mazziotti G, Cuomo G, Ressa M, Sorvillo F, Amato G et al. Hashimoto's thyroiditis is associated with peripheral lymphocyte activation in patients with systemic sclerosis. Clin Exp Rheumatol 2005; 23: 43–49.

    CAS  PubMed  Google Scholar 

  18. Gerber MA, Rapacz P, Kalter SS, Ballow M . Cell-mediated immunity in cat-scratch disease. J Allergy Clin Immunol 1986; 78 (5 Part 1): 887–890.

    Article  CAS  PubMed  Google Scholar 

  19. Chen W, Thoburn C, Hess AD . Characterization of the pathogenic autoreactive T cells in cyclosporine-induced syngeneic graft-versus-host disease. J Immunol 1998; 161: 7040–7046.

    CAS  PubMed  Google Scholar 

  20. Chetty R, Vaithilingum M, Thejpal R . Epstein–Barr virus status and the histopathological changes of parotid gland lymphoid infiltrates in HIV-positive children. Pathology 1999; 31: 413–417.

    Article  CAS  PubMed  Google Scholar 

  21. Langerak AW, Szczepanski T, Van der Burg M, Wolvers-Tettero ILM, Van Dongen JJM . Heteroduplex PCR analysis of rearranged Tcell receptor genes for clonality assessment in suspect T cell proliferations. Leukemia 1997; 11: 2192–2199.

    Article  CAS  PubMed  Google Scholar 

  22. Kneba M, Bolz I, Linke B, Hiddemann W . Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis. Blood 1995; 86: 3930–3937.

    CAS  PubMed  Google Scholar 

  23. Iijima T, Inadome Y, Noguchi M . Clonal proliferation of B lymphocytes in the germinal centers of human reactive lymph nodes: possibility of overdiagnosis of B cell clonal proliferation. Diagn Mol Pathol 2000; 9: 132–136.

    Article  CAS  PubMed  Google Scholar 

  24. Brauninger A, Yang W, Wacker HH, Rajewsky K, Kuppers R, Hansmann ML . B-cell development in progressively transformed germinal centers: similarities and differences compared with classical germinal centers and lymphocyte-predominant Hodgkin disease. Blood 2001; 97: 714–719.

    Article  CAS  PubMed  Google Scholar 

  25. Droese J, Langerak AW, Groenen PJ, Bruggemann M, Neumann P, Wolvers-Tettero IL et al. Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies. Leukemia 2004; 18: 1531–1538.

    Article  CAS  PubMed  Google Scholar 

  26. Lee MW, Lee DP, Choi JH, Moon KC, Koh JK . Failure to detect clonality in eosinophilic pustular folliculitis with follicular mucinosis. Acta Derm Venereol 2004; 84: 305–307.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Ingrid LM Wolvers-Tettero, Ellen J van Gastel-Mol, Monique ECM Oud, Brenda Verhaaf (Rotterdam), Francis Devez (Paris), Helen White (Southampton), Jonathan Langdown, Karen Brown (Cambridge), Patrícia Pontes (Porto), Kirsten Linsmeier, Nicola Passow, Cornelia Rütz, Carmen Scherer (Heidelberg), Klaas Kooistra and Jelle Conradie (Groningen) for technical assistance. The BIOMED-2 Pathology Review Panel consisted of JHJM van Krieken (Nijmegen, coordinator), F Berger (Lyon), B Jasani (Cardiff), D Canioni (Paris), PhM Kluin (Groningen), JM Cabeçadas (Lisboa), TJ Molina (Paris), G Delsol (Toulouse), A Orfao (Salamanca), JF Garcia (Madrid), M Ott (Würzburg), P Gaulard (Creteil), ST Pals (Amsterdam) and ML Hannsmann (Frankfurt). This study was supported by the BIOMED-2 Concerted Action grant BMH4-CT98-3936 of the European Commission. TJM was supported by a ‘Ligue contre le cancer, Comité de Paris’ equipment grant. FLL was supported by a grant from the Leukaemia Research Fund, UK.

Author information

Authors and Affiliations

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

    A W Langerak & J J M van Dongen

  2. Department of Pathology, Hospital Hotel-Dieu de Paris, Paris, France

    T J Molina

  3. Wessex Immunology Service, Molecular Pathology Unit, Southampton University Hospitals, Southampton, UK

    F L Lavender

  4. Department of Pathology, Molecular Histopathology, Addenbrooke's NHS Trust Hospital, Cambridge, UK

    D Pearson

  5. Department of Human Genetics, University of Heidelberg, Heidelberg, Germany

    T Flohr

  6. Department of Pathology, University of Porto and IPATIMUP (Institute for Molecular Pathology and Immunology of Porto University), Porto, Portugal

    C Sambade

  7. Department of Pathology, University Medical Center Groningen, Groningen, The Netherlands

    E Schuuring

  8. Department of Pathology, Hopital Purpan, Toulouse, France

    T Al Saati

  9. Department of Pathology, UMC St Radboud, Nijmegen, The Netherlands

    J H J M van Krieken

Authors
  1. A W Langerak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T J Molina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F L Lavender
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Pearson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T Flohr
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C Sambade
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E Schuuring
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. T Al Saati
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J J M van Dongen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J H J M van Krieken
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J J M van Dongen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Langerak, A., Molina, T., Lavender, F. et al. Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 21, 222–229 (2007). https://doi.org/10.1038/sj.leu.2404482

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2404482

Keywords

This article is cited by

Search

Advanced search

Quick links