Molecular Medicine

, Volume 19, Issue 1, pp 230–236 | Cite as

Temporal Dynamics of Clonal Evolution in Chronic Lymphocytic Leukemia with Stereotyped IGHV4-34/IGKV2-30 Antigen Receptors: Longitudinal Immunogenetic Evidence

  • Lesley-Ann Sutton
  • Efterpi Kostareli
  • Evangelia Stalika
  • Athanasios Tsaftaris
  • Achilles Anagnostopoulos
  • Nikos Darzentas
  • Richard Rosenquist
  • Kostas Stamatopoulos
Research Article


Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset 4 possess distinctive patterns of intraclonal diversification (ID) within their immunoglobulin (IG) genes. Although highly indicative of an ongoing response to antigen(s), the critical question concerning the precise timing of antigen involvement is unresolved. Hence, we conducted a large-scale longitudinal study of eight subset 4 cases totaling 511 and 398 subcloned IG heavy and kappa sequences. Importantly, we could establish a hierarchical pattern of subclonal evolution, thus revealing which somatic hypermutations were negatively or positively selected. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially; however, at later time points, the minor cluster had often disappeared and hence not been selected. Despite the high intensity of ID, it was remarkable that certain residues remained essentially unaltered. These novel findings strongly support a role for persistent antigen stimulation in the clonal evolution of CLL subset 4.



This work was supported in part by the ENosAI project (code 09SYN-13-880), cofunded by the European Union and the Hellenic General Secretariat for Research and Technology, to N Darzentas and K Stamatopoulos; the Cariplo Foundation (Milan, Italy) to K Stamatopoulos; and the Swedish Cancer Society, the Swedish Research Council, and the Lion’s Cancer Research Foundation, Uppsala. N Darzentas is currently supported by Central European Institute of Technology, Masaryk University (CEITEC MU) (CZ.1.05/1.1.00/02.0068) and SuPReMMe (CZ.1.07/2.3.00/20.0045).

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  1. 1.
    Schroeder HW Jr, Dighiero G. (1994) The pathogenesis of chronic lymphocytic leukemia: analysis of the antibody repertoire. Immunol. Today. 15:288–94.CrossRefPubMedGoogle Scholar
  2. 2.
    Hashimoto S, et al. (1995) Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells. J. Exp. Med. 181:1507–17.CrossRefPubMedGoogle Scholar
  3. 3.
    Efremov DG, et al. (1996) Restricted immunoglobulin VH region repertoire in chronic lymphocytic leukemia patients with autoimmune hemolytic anemia. Blood. 87:3869–76.PubMedGoogle Scholar
  4. 4.
    Johnson TA, Rassenti LZ, Kipps TJ. (1997) Ig VH1 genes expressed in B cell chronic lymphocytic leukemia exhibit distinctive molecular features. J. Immunol. 158:235–46.PubMedGoogle Scholar
  5. 5.
    Fais F, et al. (1998) Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J. Clin. Invest. 102:1515–25.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Chiorazzi N, Ferrarini M. (2003) B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor. Annu. Rev. Immunol. 21:841–94.CrossRefPubMedGoogle Scholar
  7. 7.
    Damle RN, et al. (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood. 94:1840–7.Google Scholar
  8. 8.
    Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. (1999) Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood. 94:1848–54.Google Scholar
  9. 9.
    Tobin G, et al. (2003) Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vlambda2-14 gene use and homologous CDR3s: implicating recognition of a common antigen epitope. Blood. 101:4952–7.CrossRefPubMedGoogle Scholar
  10. 10.
    Ghiotto F, et al. (2004) Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia. J. Clin. Invest. 113:1008–16.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Widhopf GF 2nd, et al. (2004) Chronic lymphocytic leukemia B cells of more than 1% of patients express virtually identical immunoglobulins. Blood. 104:2499–504.CrossRefPubMedGoogle Scholar
  12. 12.
    Messmer BT, et al. (2004) Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. J. Exp. Med. 200:519–25.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tobin G, et al. (2004) Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia. Blood. 104:2879–85.CrossRefPubMedGoogle Scholar
  14. 14.
    Ghia P, et al. (2005) Geographic patterns and pathogenetic implications of IGHV gene usage in chronic lymphocytic leukemia: the lesson of the IGHV3-21 gene. Blood. 105:78–1685.CrossRefGoogle Scholar
  15. 15.
    Thorselius M, et al. (2006) Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status. Blood. 107:2889–94.CrossRefPubMedGoogle Scholar
  16. 16.
    Stamatopoulos K, et al. (2007) Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenetic implications and clinical correlations. Blood. 109:259–70.CrossRefPubMedGoogle Scholar
  17. 17.
    Murray F, et al. (2008) Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis. Blood. 111:1524–33.CrossRefPubMedGoogle Scholar
  18. 18.
    Darzentas N, et al. (2010) A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence. Leukemia. 24:125–32.CrossRefPubMedGoogle Scholar
  19. 19.
    Agathangelidis A, et al. (2012) Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies. Blood. 119:4467–75.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bomben R, et al. (2009) Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: results from an Italian multicentre study. Br. J. Haematol. 144:492–506.CrossRefPubMedGoogle Scholar
  21. 21.
    Tobin G, et al. (2002) Somatically mutated Ig V(H)3-21 genes characterize a new subset of chronic lymphocytic leukemia. Blood. 99:2262–4.CrossRefPubMedGoogle Scholar
  22. 22.
    Strefford JC, et al. (2013) Distinct patterns of novel gene mutations in poor-prognostic stereotyped subsets of chronic lymphocytic leukemia: the case of SF3B1 and subset #2. Leukemia. 2013, May 3 [Epub ahead of print].Google Scholar
  23. 23.
    Lanemo Myhrinder A et al. (2008) A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies. Blood. 111:3838–48.CrossRefPubMedGoogle Scholar
  24. 24.
    Catera R, et al. (2008) Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation. Mol. Med. 14:665–74.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zwick C, et al. (2013) Autoantigenic targets of B-cell receptors derived from chronic lymphocytic leukemias bind to and induce proliferation of leukemic cells. Blood 121:4708–17.CrossRefPubMedGoogle Scholar
  26. 26.
    Herve M, et al. (2005) Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity. J. Clin. Invest. 115:1636–43.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chu CC, et al. (2008) Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA. Blood. 112:5122–9.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Chu CC, et al. (2010) Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin. Blood 115:3907–15.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Sutton LA, et al. (2009) Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen. Blood. 114:4460–8.CrossRefGoogle Scholar
  30. 30.
    Kostareli E, et al. (2010) Intraclonal diversification of immunoglobulin light chains in a subset of chronic lymphocytic leukemia alludes to antigen-driven clonal evolution. Leukemia. 24:1317–24.CrossRefGoogle Scholar
  31. 31.
    Hadzidimitriou A, et al. (2009) Evidence for the significant role of immunoglobulin light chains in antigen recognition and selection in chronic lymphocytic leukemia. Blood. 113:403–11.CrossRefGoogle Scholar
  32. 32.
    Hallek M, et al. (2008) Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 111:5446–56.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Lefranc MP, et al. (2009) IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 37:D1006–12.CrossRefPubMedGoogle Scholar
  34. 34.
    Brochet X, Lefranc MP, Giudicelli V. (2008) IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 36:W503–8.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Krishnan MR, Jou NT, Marion TN. (1996) Correlation between the amino acid position of arginine in VH-CDR3 and specificity for native DNA among autoimmune antibodies. J. Immunol. 157:2430–9.PubMedGoogle Scholar
  36. 36.
    Li Z, Schettino EW, Padlan EA, Ikematsu H, Casali P. (2000) Structure-function analysis of a lupus anti-DNA autoantibody: central role of the heavy chain complementarity-determining region 3 Arg in binding of double- and single-stranded DNA. Eur. J. Immunol. 30:2015–26.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Jang YJ, Stollar BD. (2003) Anti-DNA antibodies: aspects of structure and pathogenicity. Cell Mol. Life Sci. 60:309–20.CrossRefPubMedGoogle Scholar
  38. 38.
    Pugh-Bernard AE, et al. (2001) Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J. Clin. Invest. 108:1061–70.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Cappione AJ, Pugh-Bernard AE, Anolik JH, Sanz I. (2004) Lupus IgG VH4.34 antibodies bind to a 220-kDa glycoform of CD45/B220 on the surface of human B lymphocytes. J. Immunol. 172:4298–307.CrossRefPubMedGoogle Scholar
  40. 40.
    Cocca BA, et al. (2001) Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells. Proc. Natl. Acad. Sci. U. S. A. 98:13826–31.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Li Y, Li H, Ni D, Weigert M. (2002) Anti-DNA B cells in MRL/lpr mice show altered differentiation and editing pattern. J. Exp. Med. 196:1543–52.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Behrendt M, et al. (2003) The role of somatic mutation in determining the affinity of anti-DNA antibodies. Clin. Exp. Immunol. 131:182–9.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Muzio M, et al. (2008) Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy. Blood. 112:188–95.CrossRefPubMedGoogle Scholar
  44. 44.
    Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. (2011) B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 118:4313–20.CrossRefPubMedGoogle Scholar
  45. 45.
    Silberstein LE, George A, Durdik JM, Kipps TJ. (1996) The V4-34 encoded anti-i autoantibodies recognize a large subset of human and mouse B-cells. Blood Cells Mol. Dis. 22:126–38.CrossRefPubMedGoogle Scholar
  46. 46.
    Potter KN, Hobby P, Klijn S, Stevenson FK, Sutton BJ. (2002) Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen. J. Immunol. 169:3777–82.CrossRefPubMedGoogle Scholar
  47. 47.
    Kostareli E, et al. (2009) Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors. Leukemia. 23:919–24.CrossRefPubMedGoogle Scholar
  48. 48.
    Rossi D, et al. (2009) Stereotyped B-cell receptor is an independent risk factor of chronic lymphocytic leukemia transformation to Richter syndrome. Clin. Cancer Res. 15:4415–22.CrossRefPubMedGoogle Scholar
  49. 49.
    Martin-Subero JI, et al. (2007) A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation. Leukemia. 21:1532–44.CrossRefPubMedGoogle Scholar
  50. 50.
    Athanasiadou A, et al. (2008) Recurrent cytogenetic findings in subsets of patients with chronic lymphocytic leukemia expressing IgG-switched stereotyped immunoglobulins. Haematologica. 93:473–4.CrossRefPubMedGoogle Scholar
  51. 51.
    Chapiro E, et al. (2008) The most frequent t(14;19)(q32;q13)-positive B-cell malignancy corresponds to an aggressive subgroup of atypical chronic lymphocytic leukemia. Leukemia. 22:2123–7.CrossRefPubMedGoogle Scholar
  52. 52.
    Marincevic M, et al. (2010) High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with ‘stereotyped’ IGHV3-21 and IGHV4-34 B-cell receptors. Haematologica. 95:1519–25.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Rossi D, et al. (2013) Association between molecular lesions and specific B-cell receptor subsets in chronic lymphocytic leukemia. Blood. 121:4902–5.CrossRefPubMedGoogle Scholar
  54. 54.
    Gounari M, et al. (2012) Promiscuous antigen reactivity may underlie clinical aggressiveness and increased risk for Richter’s syndrome in chronic lymphocytic leukemia with stereotyped IGHV4-39/IGKV1(D)-39 B cell receptors. Oral session presented at: 54th ASH Annual Meeting and Exposition; 2012 Dec 8–11; Atlanta, GA ASH Abstract.Google Scholar
  55. 55.
    Ntoufa S, et al. (2012) Distinct innate immunity pathways to activation and tolerance in subgroups of chronic lymphocytic leukemia with distinct immunoglobulin receptors. Mol. Med. 18:1281–91.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Colombo M, et al. (2011) Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia. Mol. Med. 17:834–9.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Duty JA, et al. (2009) Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors. J. Exp. Med. 206:139–51.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Andrews SF, Wilson PC. (2010) The anergic B cell. Blood. 115:4976–8.CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  • Lesley-Ann Sutton
    • 1
  • Efterpi Kostareli
    • 2
  • Evangelia Stalika
    • 2
    • 3
  • Athanasios Tsaftaris
    • 3
  • Achilles Anagnostopoulos
    • 2
  • Nikos Darzentas
    • 3
    • 4
  • Richard Rosenquist
    • 1
  • Kostas Stamatopoulos
    • 2
    • 3
  1. 1.Department of Immunology, Genetics and Pathology, Rudbeck LaboratoryUppsala UniversityUppsalaSweden
  2. 2.Hematology Department and HCT UnitThessalonikiGreece
  3. 3.Institute of Applied BiosciencesCenter for Research and Technology Hellas (CERTH)ThessalonikiGreece
  4. 4.Molecular Medicine Program, Central European Institute of TechnologyMasaryk UniversityBrnoCzech Republic

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