Malleable Immunoglobulin Genes and Hematopathology – The Good, the Bad, and the Ugly
2008; Elsevier BV; Volume: 10; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2008.080061
ISSN1943-7811
Autores Tópico(s)Platelet Disorders and Treatments
ResumoImmunoglobulin gene rearrangement analysis is one of the more commonly performed assays available on the hematopathology menu of clinical molecular pathology laboratories. The analysis of these rearrangements provides useful information on a number of different levels in the evaluation of lymphoproliferations. An appreciation of the various mechanisms involved in the numerous physiological pathways affecting the immunoglobulin genes, and hence antibody molecules, is central to an understanding of B-cell development vis-à-vis the generation of immunological diversity. Knowledge about the intricate complexities of these mechanisms is also germane to an evaluation of testing methodologies. With this information, it is easier to develop an understanding of how contemporary molecular testing of immunoglobulin gene rearrangements has evolved, from historically quite heterogeneous, fairly flawed, and rather ugly approaches to current more-standardized protocols. In addition, recognition of how such genetic changes with good intentions can turn bad has fostered increasing insights into the pathogenesis of B-cell lymphomas and leukemias. Despite the significant improvements in the design of immunoglobulin gene rearrangement assays, numerous pitfalls and caveats remain. Accordingly, it is crucial to consider such testing a tool, and although most useful, it is one of many tools that may be required to build cogent diagnoses. Immunoglobulin gene rearrangement analysis is one of the more commonly performed assays available on the hematopathology menu of clinical molecular pathology laboratories. The analysis of these rearrangements provides useful information on a number of different levels in the evaluation of lymphoproliferations. An appreciation of the various mechanisms involved in the numerous physiological pathways affecting the immunoglobulin genes, and hence antibody molecules, is central to an understanding of B-cell development vis-à-vis the generation of immunological diversity. Knowledge about the intricate complexities of these mechanisms is also germane to an evaluation of testing methodologies. With this information, it is easier to develop an understanding of how contemporary molecular testing of immunoglobulin gene rearrangements has evolved, from historically quite heterogeneous, fairly flawed, and rather ugly approaches to current more-standardized protocols. In addition, recognition of how such genetic changes with good intentions can turn bad has fostered increasing insights into the pathogenesis of B-cell lymphomas and leukemias. Despite the significant improvements in the design of immunoglobulin gene rearrangement assays, numerous pitfalls and caveats remain. Accordingly, it is crucial to consider such testing a tool, and although most useful, it is one of many tools that may be required to build cogent diagnoses. Molecular genetic testing is an integral facet of the evaluation of hematological disorders, in particular in the work-up of hematological neoplasms.1Paessler ME Bagg A Use of molecular techniques in the analysis of hematologic diseases.in: Hoffman R Shattil SJ Furie B Cohen HJ Silberstein LE McGlave P Strauss M Benz EJ Hematology: Basic Principles and Practice. ed 4. Elsevier, Philadelphia2005: 2713-2726Google Scholar This includes not only the ability to characterize specific leukemias and lymphomas but also to distinguish reactive from neoplastic disorders both lymphoproliferations and, more recently, myeloproliferations.2Tefferi A Vardiman JW Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms.Leukemia. 2008; 22: 14-22Crossref PubMed Scopus (836) Google Scholar Allied to its diagnostic utility, molecular analysis is central to contemporary classification and prognostic assignment,3Leich E Hartmann EM Burek C Ott G Rosenwald A Diagnostic and prognostic significance of gene expression profiling in lymphomas.APMIS. 2007; 115: 1135-1146Crossref PubMed Scopus (21) Google Scholar,4Bagg A Role of molecular studies in the classification of lymphoma.Expert Rev Mol Diagn. 2004; 4: 83-97Crossref PubMed Scopus (11) Google Scholar and it is particularly well suited to testing for minimal residual disease after therapy.5Szczepański T Why and how to quantify minimal residual disease in acute lymphoblastic leukemia?.Leukemia. 2007; 21: 622-626PubMed Google Scholar,6Ou J Vergilio JA Bagg A Molecular diagnosis and monitoring in the clinical management of patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors.Am J Hematol. 2008; 83: 296-302Crossref PubMed Scopus (26) Google Scholar A variety of molecular phenomena are amenable to routine diagnostic molecular analysis, with an assessment of gene rearrangements currently being the most commonly evaluated phenomenon. Gene rearrangements can be conveniently grouped into two broad categories: physiological and pathological. Physiological gene rearrangements refer to the normal intragenic shuffling of segments of antigen receptor genes, namely immunoglobulin (IG) genes and T-cell receptor (TCR) genes in B and T cells, respectively, that represent a major mechanism in the generation of immunological diversity. By contrast, pathological rearrangements, which are essentially synonymous with chromosomal translocations or inversions, lead to the movement of genes that are physiologically kept separate (ie, this is an intergenic phenomenon). Translocations lead to one of two major consequences that can be considered to have either a qualitative or quantitative effect. Those translocations that cause the disruption of genes, with the subsequent fusion of portions of the disrupted genes, resulting in the generation of a novel, pathological chimeric gene and ultimately chimeric oncoprotein, can be considered qualitative. There are numerous well-characterized examples, which include the BCR-ABL1 fusion generated as a consequence of the t(9;22) in chronic myelogenous leukemia and a subset of (in particular, adult) precursor B-cell acute lymphoblastic leukemia, and the NPM1-ALK fusion/t(2;5) found in some cases of anaplastic large-cell lymphoma. By contrast, a translocation resulting in the inappropriate overexpression of an intact gene (which physiologically typically has its expression tightly regulated), often due to the apposition of enhancers or promoters of contextually highly expressed genes, can be considered quantitative. Here, too, there are many well-characterized and recognized examples, such as the overexpression of BCL2 and CCND1 as a consequence of being juxtaposed with immunoglobulin heavy chain gene (IGH@) in the t(14;18) and t(11;14) associated with follicular and mantle cell lymphoma, respectively. However, this heightened expression is not always a consequence of positive regulation, in that removal of negative regulatory elements may also be operative, as has been demonstrated with translocations involving the LMO2 gene in T-cell acute lymphoblastic leukemia.7Dik WA Nadel B Przybylski GK Asnafi V Grabarczyk P Navarro JM Verhaaf B Schmidt CA Macintyre EA van Dongen JJ Langerak AW Different chromosomal breakpoints impact the level of LMO2 expression in T-ALL.Blood. 2007; 110: 388-392Crossref PubMed Scopus (42) Google Scholar In general terms, the diagnostically relevant end point of analyzing physiological gene rearrangements compared with pathological rearrangements is quite different. For antigen receptor gene rearrangements, the major determination is whether there is homogeneity versus heterogeneity, essentially translating into monoclonality versus polyclonality, which may then be interpreted, with numerous caveats—discussed below, as neoplastic versus reactive. By contrast, the end point of an assessment of diagnostic pathological rearrangements using standard qualitative PCR assays is somewhat different, requiring either a positive (present) or negative (absent) readout. However, such qualitative absolutes, of positive versus negative, although pertinent in the diagnostic setting, are not applicable in the posttherapeutic scenario, where more sensitive and precise quantitative measurements are clearly important. There are two broad scenarios in which an analysis of antigen receptor gene rearrangements (ARGRs) in general and IG rearrangements in particular can be considered: for initial diagnosis and for subsequent minimal residual disease studies (Table 1). In the diagnostic setting, when evaluating lymphoid tissue microscopically and when the tissue is qualitatively and quantitatively optimal, it is usually quite straightforward to distinguish neoplastic from reactive disorders, in most instances. On such occasions, it is not necessary to perform ARGR analysis for diagnostic purposes. However, in a minor subset of cases, the histomorphological features (and immunophenotypic findings) may not be unequivocal, and it is in these scenarios that ARGR studies may be particularly helpful in providing an assessment of clonality. The frequency with which such cases require clonality testing by ARGR analyses is difficult to define but has been estimated to be required in as many as ∼30% of cases in laboratories with limited specialization in hematopathology and ∼10% of cases in specialized hematopathology centers.8van Krieken JH Langerak AW Macintyre EA Kneba M Hodges E Sanz RG Morgan GJ Parreira A Molina TJ Cabeçadas J Gaulard P Jasani B Garcia JF Ott M Hannsmann ML Berger F Hummel M Davi F Brüggemann M Lavender FL Schuuring E Evans PA White H Salles G Groenen PJ Gameiro P Pott Ch Dongen JJ Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.Leukemia. 2007; 21: 201-206Crossref PubMed Scopus (251) Google Scholar In addition, with the use of less-invasive diagnostic procedures such as fine needle aspiration and thin needle core biopsies, pathologists are being called on to render diagnoses on lesser amounts of tissue. Although immunophenotypic analysis here is often helpful, the inability to assess architecture, an important facet in the evaluation of lymphoid tissue, may compromise the ability to render a specific or even general (neoplastic versus reactive) diagnosis. An evaluation of ARGR in these situations can provide diagnostically useful information. These studies may also be of value in establishing (or excluding) clonal relationships in two distinct lymphomas that might be separated anatomically and/or chronologically. For example, they may answer such questions as the following: Are the two lymphomas with which the patient presented clonally related? Is this recurrent disease, or a new and different lymphoma? Does this large-cell lymphoma reflect transformation of the small-cell lymphoma?Table 1Possible Indications for Performing Antigen Receptor Gene Rearrangement AnalysisScenarioCommentDiagnosis Neoplastic versus reactiveAtypical lymphoproliferations in which the lesion is neither definitively neoplastic nor reactive by histomorphology and immunophenotypic analysis Limited tissueSmall needle biopsies and fine needle aspirations, precluding the ability to evaluate architecture Evaluating clonal relationshipsIn different tumors that are separated anatomically and/or chronologically Equivocal immunophenotypeNumerous caveats to using molecular studies to assign lineage; thus, use this application extremely judiciously Atypical T-cell lymphoproliferationsBecause there is not a similarly useful immunophenotypic marker of clonality as there is for mature B cells (κ:λ ratio)Minimal residual disease Response to therapyThe rate of clearance of disease may be prognostic Early recurrenceAfter attainment of remission Product contaminationFor autologous transplants Increased precursor B cellsIn patients with precursor B-cell neoplasms, to distinguish residual disease from hematogones Open table in a new tab A fourth context in which ARGR analysis might theoretically be valuable is in the assignment of lineage, when immunophenotypic studies are unhelpful. However, this use of ARGR studies is fraught with caveats and is to be avoided because neoplastic (and sometimes physiological) lymphoid cells can evince cross lineage rearrangements, ie, IG gene rearrangements in T cells and TCR gene rearrangements in B cells.9Bagg A Immunoglobulin and T-cell receptor gene rearrangements: minding your B's and T's in assessing lineage and clonality in neoplastic lymphoproliferative disorders.J Mol Diagn. 2006; 8: 426-429Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Furthermore, with the development of more robust immunophenotypic assays, particularly reagents that are applicable to fixed tissue, situations in which they are unhelpful are less commonly encountered now. Nevertheless, there are scenarios where the judicious use of these studies may be helpful. For example, there is a normal hierarchy of IG gene rearrangements, with IGH@ rearranging before the light chains do in normal B-cell ontogeny. Thus, although IGH@ gene rearrangements are not uncommon in T-cell malignancies, the finding of a light chain gene rearrangement is less common in these cells, and finding an immunoglobulin light chain gene rearrangement is more likely to reflect the commitment to bona fide B-cell, rather than T-cell, lineage; however, this is certainly not absolute.10van Dongen JJ Langerak AW Brüggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E García-Sanz R van Krieken JH Droese J González D Bastard C White HE Spaargaren M González M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA 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: 2257-2317Crossref PubMed Scopus (2507) Google Scholar Finally, although neoplastic B-cell disorders significantly outnumber neoplastic T-cell disorders, particularly in the Western hemisphere (in a ratio of ∼6:1), a molecular diagnostic laboratory is perhaps more likely to be called on to perform TCR gene rearrangement studies compared with IG gene rearrangement analyses. The reason for this is that it is usually possible to assign clonality in (mature) B cells by immunophenotypic, in particular flow cytometric, analysis via an evaluation of κ- and λ-immunoglobulin light chain expression, whereas no similarly useful, simple, and widely available immunophenotypic method of clonality assessment exists for T cells. (There are flow cytometric assays that might facilitate this, by assessing the use of different TCR β chain families, but these approaches are more complex than κ- versus λ-analyses.11Morice WG Kimlinger T Katzmann JA Lust JA Heimgartner PJ Halling KC Hanson CA Flow cytometric assessment of TCR-Vbeta expression in the evaluation of peripheral blood involvement by T-cell lymphoproliferative disorders: a comparison with conventional T-cell immunophenotyping and molecular genetic techniques.Am J Clin Pathol. 2004; 121: 373-383Crossref PubMed Scopus (92) Google Scholar,12Beck RC Stahl S O'Keefe CL Maciejewski JP Theil KS Hsi ED Detection of mature T-cell leukemias by flow cytometry using anti-T-cell receptor V beta antibodies.Am J Clin Pathol. 2003; 120: 785-794Crossref PubMed Scopus (58) Google Scholar) In addition to the above diagnostic scenarios, an assessment of ARGR can be particularly useful in the assessment of minimal residual disease (MRD), when extremely sensitive PCR-based approaches can detect neoplasia well below the level that can be appreciated morphologically (and sometimes immunophenotypically). Thus, for patients in whom therapy is given with curative intent and/or in whom the measurement of MRD is clinically relevant (ie, predictive of outcome, with the potential for meaningful therapeutic intervention), it is useful to evaluate a diagnostic specimen for its specific ARGR(s). This is performed not necessarily for primary diagnostic purposes but rather to provide a molecular fingerprint that can be used for subsequent tracking of MRD. There are a number of somewhat distinct settings in which MRD assessment can be performed and may be clinically useful. This includes the initial phases of chemotherapy for acute lymphoblastic leukemia, in which the ability to reduce the level of disease below a certain threshold (typically of the order of a 4-log reduction from diagnosis) is considered an extremely favorable prognostic variable. By contrast, cases in which there is a less than 2 log reduction, reflective of possible chemotherapy resistance, fare much more poorly and may be considered for alternative therapeutic modalities.13van der Velden VH Panzer-Grümayer ER Cazzaniga G Flohr T Sutton R Schrauder A Basso G Schrappe M Wijkhuijs JM Konrad M Bartram CR Masera G Biondi A van Dongen JJ Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting.Leukemia. 2007; 21: 706-713PubMed Google Scholar A second context in which MRD testing may be of value is in monitoring patients after remission, including molecular remission, has been attained to assess for early relapse.14Raff T Gökbuget N Lüschen S Reutzel R Ritgen M Irmer S Böttcher S Horst HA Kneba M Hoelzer D Brüggemann M GMALL Study Group Molecular relapse in adult standard-risk ALL patients detected by prospective MRD monitoring during and after maintenance treatment: data from the GMALL 06/99 and 07/03 trials.Blood. 2007; 109: 910-915Crossref PubMed Scopus (183) Google Scholar Therapeutic intervention here, when the tumor burden is relatively low, may be more effective than when attempting to treat at the time of florid hematological or clinical relapse, as has been demonstrated in some myeloid leukemias.15Cervetti G Galimberti S Andreazzoli F Fazzi R Cecconi N Caracciolo F Petrini M Rituximab as treatment for minimal residual disease in hairy cell leukaemia.Eur J Haematol. 2004; 73: 412-417Crossref PubMed Scopus (54) Google Scholar,16Kim YJ Kim DW Lee S Chung NG Hwang JY Kim YL Min CK Kim CC Preemptive treatment of minimal residual disease post transplant in CML using real-time quantitative RT-PCR: a prospective, randomized trial.Bone Marrow Transplant. 2004; 33: 535-542Crossref PubMed Scopus (9) Google Scholar Third, MRD testing can be applied to stem cell products that are to be used for autologous transplantation, to ensure that the reinfused material is free of contaminating tumor.17Corradini P Carrabba MG Farina L Molecular methods used for the detection of autologous graft contamination in lymphoid disorders.Methods Mol Med. 2007; 134: 179-196Crossref PubMed Scopus (1) Google Scholar Finally, in addition to the above three contexts in which MRD testing exploits the sensitivity of testing, the specificity of ARGR can also be a valuable phenomenon. This refers to patients with precursor B-cell lymphoblastic leukemias, in which in posttherapy bone marrow specimens, it may be challenging to distinguish physiological, regenerating precursor B cells (hematogones) from neoplastic precursor B cells (residual disease). Although flow cytometric analysis ought to be able to make this distinction, a simple PCR for ARGR may be just as informative and potentially more specific.18Kallakury BV Hartmann DP Cossman J Gootenberg JE Bagg A Post-therapy surveillance of B-cell precursor acute lymphoblastic leukemia: the value of polymerase chain reaction and limitations of flow cytometry.Am J Clin Pathol. 1999; 111: 759-766PubMed Google Scholar Importantly, however, the family-specific primers used for qualitative diagnostic testing (see Evolution of Testing Methodologies to Assess Immunoglobulin Gene Rearrangements) do not have sufficient sensitivity to allow for meaningful MRD testing, in that they are able to detect only ∼5 to 10% clonal cells. By contrast, for MRD measurements to be clinically relevant, sensitivities need to be achieved on the order of 0.01%, levels that cannot be attained with the primers used in diagnostic testing. Accordingly, analysis must be performed by quantitative (typically real-time) PCR, using reagents (primers and/or probes) that are specific for the IGH@ gene rearrangement present in the B-cell neoplasm that is being monitored. The production of such reagents is laborious, requiring the cloning and sequencing of DNA sequences in each individual case, in and around the region of the IGH@ gene that is essentially specific for each B cell and hence each B-cell neoplasm. Any understanding of the utility (and pitfalls) of clinical diagnostic IG analysis is predicated on an understanding of the physiological mechanisms involved in the generation of diverse IG genes and ultimately Ig molecules (antibodies). Similar mechanisms underlie the generation of the diversity of T-cell repertoire, via the generation of numerous T-cell receptors, the analysis of which is equally invaluable in the determination of clonality of T cells; however, the focus of this discussion is B cells and their IG genes. It is perhaps not too oversimplified to consider that the raison d'etre of a B cell is to generate a functional and specific antibody molecule. Collectively, B cells are called on to generate a plethora of antibodies to account for all possible foreign antigens to which the human organism might be exposed. However, there are insufficient genes in the whole human genome (never mind within the IG genes themselves) to account for the vast number of antibodies required. Thus, there are a number of different mechanisms that allow for the creation of a multitude of different IG genes and ultimately antibody molecules (Figure 1). These mechanisms include combinatorial diversity, junctional diversity, somatic hypermutation, and class switch recombination. They are mediated by a variety of key enzymes, including recombinase-activating genes (RAG) 1 and 2, terminal deoxynucleotidyl transferase, and activation-induced cytosine deaminase. The first step in the generation of diversity occurs very early in B-cell differentiation, while it is still developing in the bone marrow, at the pro B-cell stage of maturation.19van Zelm MC van der Burg M de Ridder D Barendregt BH de Haas EF Reinders MJ Lankester AC Révész T Staal FJ van Dongen JJ Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression.J Immunol. 2005; 175: 5912-5922Crossref PubMed Scopus (137) Google Scholar Importantly, this apparently stochastic rearrangement of disparate segments within the IG genes is antigen independent, occurring long before exposure to antigen; the B cell at this stage, lacking the ability to synthesize and express Ig molecules, is not yet equipped to even recognize antigens. In the germline of such B cells, the IGH@ gene contains numerous disparate V (variable), D (diversity), and J (joining) gene segments. All other cells, of course, contain these gene segments in the germline configuration, too; however, their subsequent rearrangement is essentially exclusive to B cells. There are ∼38 to 46 functional V segments (there are many more pseudo and rearrangeable, but nonfunctional V segments), ∼23 D segments, and 6 J segments [Immunogentics Repertoire (IG and TR); http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/locusdesc/human/IGH/Hu_IGHdesc.html, last accessed April 28, 2008] with one segment from each of these three different regions randomly recombining to generate a specific VDJ gene rearrangement. Flanking the V, D, and J segments are recombination signal sequences, consisting of conserved heptamers and nonamers, separated by nonconserved spacers, typically of 12 or 23 nucleotides. These recombination signal sequences are important in ensuring legitimate recombination, and they provide binding sites for the primary enzyme complex (recombinase activating genes RAG1/RAG2) that mediates the initial DNA cleavage and synapsis required for this event. A variety of other DNA repair factors are involved in this reaction, including DNA-PKc, Ku70, Ku80, Artemis, DNA ligase IV, XRCC4, and Cernunnos. There is a hierarchy both in the order of IG gene rearrangements at the different loci (IGH@ on 14q32, then IGK@ on 2p11, and then IGL@ on 22q11) and within the IGH@ locus. Thus, with regard to the IGH@ locus, DJ rearrangement occurs first, and only once this is completed is V to DJ rearrangement able to occur.20Kipps TJ Human B cell biology.Int Rev Immunol. 1997; 15: 243-264Crossref PubMed Scopus (12) Google Scholar In addition to this important apparently random shuffling mechanism for the generation of immunological diversity (so called combinatorial diversity), nucleotides are also randomly both deleted and added at the sites of V to D and D to J fusion. This process is primarily mediated by the enzyme terminal deoxynucleotidyl transferase (TdT) and is referred to as generating junctional diversity, with the added nucleotides creating N regions between V and D and between D and J. Additional mechanisms contribute to junctional diversity, such as the inclusion of P (for pallindromic) nucleotides by RAG1/2 and the exonuclease activity of the DNA repair machinery. Based on the number of functional IGH@ V, D, and J segments, there are on the order of 104 possible recombination events; together with VJ rearrangements at each of the IGK@ and IGL@ loci (both of which lack D segments), a total of ∼106 different antibody molecules can be yielded via combinatorial mechanisms.21Janeway C Immunobiology.in: The Immune System in Health and Disease. ed 6. Garland Science, New York2005Google Scholar Junctional diversity (through the actions of both TdT and the other noted mechanisms) is responsible for at least 6 additional orders of magnitude of diversity, thus allowing for the potential of >1012 different antibody molecules.22Sagaert X Sprangers B De Wolf-Peeters C The dynamics of the B follicle: understanding the normal counterpart of B-cell-derived malignancies.Leukemia. 2007; 21: 1378-1386Crossref PubMed Scopus (20) Google Scholar Thus, two major and temporally related diversification mechanisms, combinatorial and junctional, mediated by RAG1/RAG2 and TdT, respectively, occur in the early bone marrow phase of B-cell development, before antigen exposure. The fully rearranged IGH@ gene (VNDNJ) contains a number of complementarity determining regions (CDRs) and framework regions (FRs) (Figure 2). As the name suggests, the CDRs encode for those components of the antibody molecule that are most intimately involved with antigen recognition and are the most diverse between antizhybodies and IGH@ genes. By contrast, the FRs are typically quite similar between different antibodies and IGH@ genes. An appreciation of these differences (CDRs versus FRs) is central to understanding the PCR-based detection of IGH@ gene rearrangements, as discussed under Evolution of Testing Methodologies to Assess Immunoglobulin Gene Rearrangements. There are three CDRs in the IGH@ gene, with the most distal (3′) CDR3 being the most heterogeneous, because it is affected by both recombinatorial and junctional diversification mechanisms described above. By contrast, the more proximal (5′) CDR1 and CDR2 are encoded for in the germline (as are all of the FRs) and are not affected by either recombination or the action of TdT. The CDRs are flanked by the total of four FR regions, with FR1, FR2, and FR3 being encoded by the V segment and FR4 residing in the J segment. The whole D segment, flanked on both sides by the extremely heterogeneous N region, fully resides in CDR3. Successful IGH@ VDJ recombination and IG light chain gene rearrangement are required for B cells to express intact and functional Ig molecules on their surface membranes, at which stage they may exit the bone marrow, having now developed their primary or central repertoire. Although these circulating B cells are now “mature,” as evidenced by their expression of Ig molecules on surface membranes, they are still considered naïve in terms of mostly not yet having been exposed to antigen. These naïve B cells, which account for ∼60 to 70% of circulating B cells, must now migrate to lymph nodes, the spleen, and mucosa-associated lymphoid tissue to generate their secondary or peripheral repertoire. Specifically, they must relocate to specialized regions of these secondary lymphoid organs, the primary follicles. Here, via intricate and complex cellular and humoral interactions, they undergo the germinal center reaction, generating secondary follicles, from which memory B cells and plasma cells will ultimately emerge. The cellular partners in these germinal center events include T cells and antigen-presenting follicular dendritic cells. However, transiting the germinal center is not an absolute requirement for the attainment of memory, in that some (marginal zone) B cells can acquire memory in a T-cell-independent manner.23Klein U Dalla-Favera R Germinal centres: role in B-cell physiology and malignancy.Nat Rev Immunol. 2008; 8: 22-33Crossref PubMed Scopus (631) Google Scholar The Ig molecules generated by VDJ recombination in the bone marrow are of low affinity, because they are created in an apparently random fashion. For these to neutralize and clear pathogenic antigens in an efficient manner, they must acquire higher affinity for these antigens and be able to perform different effector functions. To this end, and although rather oversimplified, there are two regions within the germinal center, in which two somewhat distinct DNA breakage events occur, which are central to this fine-tuning of the humoral immune response (Figure 1).24Ramiro A San-Martin BR McBride K Jankovic M
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