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Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the...
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... This finding is somewhat unexpected since M-CLL cells are likely to have passed through germinal centers where somatic IGHV hypermutation (SHM) and isotype switching take place, although it may relate to the capacity of surface IgM to deliver more efficient tonic signals than IgG or IgA [83]. Although intra-clonal isotype switching from IgM to IgG or IgA is observed in many CLL cases, it is confined to a minor sub-clonal component, with a few exceptions, as it may not offer a special survival advantage to the leukemic cells [89,90]. Certain CLL cases express stereotypes invariably connected with an IgG isotype, such as subset #4 or #8 stereotypes, suggesting that tonic signals are substituted for by other more advantageous signals in these clones [56]. ...
The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.
... Of note, mRNA transcripts coding for CLL-specific IGHV/D/J rearrangements have been found in preswitch B cells from IgG-expressing CLL patients. 168 Furthermore, differentiation of CLL cells into Ig-secreting plasma cells can arise spontaneously in vivo 169,170 and be induced in vitro. 171,172 CELLULAR ORIGIN(S) OF CLL 1787 BLOOD, 10 FEBRUARY 2011 ⅐ VOLUME 117, NUMBER 6 ...
Several cell types have been suggested as giving rise to chronic lymphocytic leukemia (CLL), and these suggestions have reflected the sophistication of technology available at the time. Although there is no consensus as to the normal cellular counterpart(s) in the disease, an antigen-experienced B lymphocyte appears required based on surface membrane phenotypes and gene expression profiles. However, what is still unclear is whether a single or multiple normal precursors were stimulated to evolve into CLL and at what stage(s) this occurred. A unifying, parsimonious theory is that CLL clones with either mutated or unmutated IGHVs derive from marginal zone B cells. However, evidence for remarkably similar B-cell receptor amino acid sequence and striking differences in polyantigen and autoantigen-binding activity, found in some but not all CLL clones, challenge a single-cell derivation for CLL. In this Perspective, we summarize data regarding normal counterparts of CLL cells and suggest that a multistep process of leukemogenesis is important to consider when assigning a cellular origin for this disease. Finally, although available data do not definitively identify the cell(s) of origin, we offer possibilities for single- and multiple-cell origin models as straw men that can be improved on and hopefully lead to final answers to this puzzle.
... 19,20 A critical question in CLL biology is whether interaction with antigen is restricted to the progenitor cell (pre-transformation phase) or whether the putative antigen may continuously trigger the CLL clone and, perhaps, drive its evolution. [22][23][24][25][26][27] The experience from other types of B-cell malignancies suggests that useful hints for an ongoing interaction with antigen may be obtained through the study of intraclonal diversification (ID) within the IG genes expressed by the malignant clone. 28 With this in mind and through a recent comprehensive analysis of ID in rearranged IGHV genes from patients with CLL, we showed that although the majority of cases showed no or low levels of ID, an intense and, likely, functionally driven ID process was evident in selected cases, especially those belonging to subset 4. 29 On these grounds, we suggested that antigen involvement may affect not only the progenitor cells of such CLL clones but also the malignant cells themselves. ...
The study of intraclonal diversification (ID) in immunoglobulin (IG) genes offers valuable insight into the role of ongoing interactions with antigen in lymphomagenesis. We recently showed that ID in the IG heavy chain genes of patients with chronic lymphocytic leukemia (CLL) was generally limited; however, intense ID was evident in selected cases, especially those expressing stereotyped IGHV4-34 rearrangements and assigned to subset 4. Here, we report results from a large-scale subcloning study of IG light variable genes, in a total of 1008 subcloned sequences from 56 CLL cases. Multiple analogies were noted between heavy and light chains regarding the occurrence and molecular features of ID. More specifically, the impact of ID on the clonotypic light chains was generally low, with the significant exception of subset 4. Similar to the IGHV4-34 heavy chains of this subset, their partner IGKV2-30 light chains were affected by an active and precisely targeted ID process. Altogether, these findings strengthen the argument that stereotypy in subset 4 extends to stereotyped ID patterns for both heavy and light chains through persistent antigenic stimulation. Furthermore, they strongly suggest that light chains have an active role in the antigen selection process, at least for certain subsets of CLL cases.
... 29,30 The available data on ID within IG genes in CLL are limited and conflicting. Several studies demonstrate some level of ID (ranging from limited to pronounced) leading to clonal evolution, [31][32][33][34][35][36] with only one study reporting a complete lack of ID. 37 We conducted the present study to address these discrepancies and systematically explore the issue as to whether CLL cells continue to acquire hypermutations after leukemic transformation. To this end, we followed a very stringent methodology and conducted a comprehensive analysis of ID in IGHV genes of diverse mutational status from 71 patients with CLL, belonging to the common IgM/IgD variant and the rare IgG-positive variant. ...
... 29,30 The studies devoted to clarifying the possible occurrence and hence implications of ID in CLL have reached conflicting results. [31][32][33][34][35][36][37] These discrepancies may be attributed, at least in part, to differences in the sensitivity of various methods used for detection of mutations, 31-37 usage of low-fidelity Taq polymerase, 31-37 number of analyzed subcloned sequences, 34 sample size, or case selection. For instance, most studies analyzed relatively few patients [31][32][33]35,37 or even a single case. ...
... [31][32][33][34][35][36][37] These discrepancies may be attributed, at least in part, to differences in the sensitivity of various methods used for detection of mutations, 31-37 usage of low-fidelity Taq polymerase, 31-37 number of analyzed subcloned sequences, 34 sample size, or case selection. For instance, most studies analyzed relatively few patients [31][32][33]35,37 or even a single case. 36 Furthermore, an underrepresentation of certain CLL subsets with distinctive SHM patterns, namely, cases using the IGHV3-21 and IGHV4-34 genes (especially in subsets 2 or 4), was observed throughout all previous studies. ...
Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).
... 8,9 Earlier, CLL cells were considered to be static in their immunoglobulin variable gene expression and mutation load. 5,11 Evidence for CLL-cell intraclonal diversification by ongoing somatic mutation, 3,[12][13][14][15][16] heavy chain receptor editing, 17 and class switching 18 suggests CLL cells can continue to differentiate. Prior studies of CLL-cell intraclonal diversification were performed using bulk CLL-cell populations and cloned heavy chain immunoglobulin gene variable regions. ...
Somatic mutations of immunoglobulin genes characterize mature memory B cells, and intraclonal B-cell diversification is typically associated with expansion of B-cell clones with greater affinity for antigen (antigen drive). Evidence for a role of antigen in progression of intraclonal chronic lymphocytic leukemia (CLL) cell diversification in patients with mutated immunoglobulin genes has not been previously presented. We performed a single-cell analysis of immunoglobulin heavy and light chains in 6 patients with somatically mutated CLL-cell immunoglobulin genes and identified 2 patients with multiple related (oligoclonal) subgroups of CLL cells. We constructed genealogic trees of these oligoclonal CLL-cell subgroups and assessed the effects of immunoglobulin somatic mutations on the ratios of replacement and silent amino acid changes in the framework and antigen-binding regions (CDRs) of the immunoglobulin heavy and light chains from each oligoclonal CLL-cell population. In one subject, the amino acid changes were consistent with an antigen-driven progression of clonally related CLL-cell populations. In the other subject, intraclonal diversification was associated with immunoglobulin amino acid changes that would have likely lessened antigen affinity. Taken together, these studies support the hypothesis that in some CLL cases intraclonal diversification is dependent on antigen interactions with immunoglobulin receptors.
... l. The techniques for PCR amplification and cloning of rearranged IgV genes have been described previously [13]. Briefly, first-strand cDNA (1 ? ...
A longitudinal study of Ig V gene segments utilized by B cells from the cerebrospinal fluid (CSF) of two patients with multiple sclerosis (MS) was carried out using RT-PCR methodologies. One patient with a relapsing-remitting (RR)-MS was investigated at onset and at relapse, 1 year later. A patient with secondary-progressive (SP)-MS was tested 9 and 13 years after disease onset. Sequence analyses of V(H)DJ(H) segments bearing V(H)3 and V(H)4 that were obtained from Cgamma cDNA genes demonstrated a substantial proportion of shared clones in the samples taken at different times; these clones were identical or closely related, i.e. had the same third complementary determining region (CDR) of the H chain variable region gene (HCDR3) with different mutations in the V(H) segment. Collectively, these data demonstrate that in MS patients there is a strong selective pressure, which could be exerted by antigen (or autoantigen) stimulation, for the maintenance and partial diversification of certain V(H)DJ(H) Cgamma sequences.
... Consistent with this, certain CLLs are characterized by an interfollicular proliferation pattern in which the neoplastic B cells surround and sometimes colonize large reactive lymphoid follicles (58). By favoring the interaction of CLL B cells with CD4 T cells and an as yet elusive Ag(s) (20, 53, 59), the GC milieu within the secondary follicle would constitute an ideal microenvironment to foster Ig CSR, plasmacytoid differentiation as well as bcl-6 and V(D)J gene somatic hypermutation (12, 19, 20) in at least some members of the leukemic clone. In addition to actively diversifying their Ig C H gene repertoire through CSR, some CLLs intraclonally diversify their surface phenotype . ...
Chronic lymphocytic leukemia (CLL) results from the expansion of malignant CD5(+) B cells that usually express IgD and IgM. These leukemic cells can give rise in vivo to clonally related IgG(+) or IgA(+) elements. The requirements and modalities of this process remain elusive. Here we show that leukemic B cells from 14 of 20 CLLs contain the hallmarks of ongoing Ig class switch DNA recombination (CSR), including extrachromosomal switch circular DNAs and circle transcripts generated by direct S micro -->Sgamma, S micro -->Salpha, and S micro -->Sepsilon as well as sequential Sgamma-->Salpha and Sgamma-->Sepsilon CSR. Similar CLL B cells express transcripts for activation-induced cytidine deaminase, a critical component of the CSR machinery, and contain germline I(H)-C(H) and mature V(H)DJ(H)-C(H) transcripts encoded by multiple Cgamma, Calpha, and Cepsilon genes. Ongoing CSR occurs in only a fraction of the CLL clone, as only small proportions of CD5(+)CD19(+) cells express surface IgG or IgA and lack IgM and IgD. In vivo class-switching CLL B cells down-regulate switch circles and circle transcripts in vitro unless exposed to exogenous CD40 ligand and IL-4. In addition, CLL B cells that do not class switch in vivo activate the CSR machinery and secrete IgG, IgA, or IgE upon in vitro exposure to CD40 ligand and IL-4. These findings indicate that in CLL at least some members of the malignant clone actively differentiate in vivo along a pathway that induces CSR. They also suggest that this process is elicited by external stimuli, including CD40 ligand and IL-4, provided by bystander immune cells.
... The PCR technique for analysis of V(D)J rearrangements and of HCDR3 length was performed as previously described (Dono et al, 1996). A RT – PCR methodology was used to detect c-myc and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA expression by the leukaemia cells treated in different manners (Cutrona et al, 1997). ...
CD10 constitutes a favourable prognostic marker for childhood acute lymphoblastic leukaemia. Since correlations between CD10, cell cycle and apoptotic abilities were demonstrated in various cell types, we investigated whether differences existed in the cycling/apoptotic abilities of CD10-positive and CD10-negative B acute lymphoblastic leukaemia cells. Twenty-eight cases of childhood acute lymphoblastic leukaemia (mean age of 6.8 years) were subdivided into two groups according to high (17 cases, 93.2±4.5%, MRFI 211±82 CD10-positive cells) or low (11 cases, 11.5±6.2%, MRFI 10±7 CD10-negative cells) expression of CD10. CD10-positive acute lymphoblastic leukaemia cells were cycling cells with elevated c-myc levels and propensity to apoptosis, whereas CD10-negative acute lymphoblastic leukaemia cells had lower cycling capacities and c-myc levels, and were resistant to apoptosis in vitro. A close correlation between all these properties was demonstrated by the observations that the few CD10-positive cells found in the CD10-negative acute lymphoblastic leukaemia group displayed elevated c-myc and cycling capacities and were apoptosis prone. Moreover, exposure of CD10-positive acute lymphoblastic leukaemia B cells to a peptide nucleic acid anti-gene specific for the second exon of c-myc caused inhibition of c-myc expression and reduced cell cycling and apoptotic abilities as well as decreased CD10 expression.
British Journal of Cancer (2002) 86, 1776–1785. doi:10.1038/sj.bjc.6600329 www.bjcancer.com
© 2002 Cancer Research UK
... VH gene fingerprinting was established for the detection of B-cell clonalities in lymphoproliferative disorders and leukemia [9,10]. This approach allows the detection of predominant clones within a panel of differently rearranged immunoglobulin genes and the identification of clonally-related genes in different samples. ...
In rheumatic diseases, autoantibody-producing cells of interest are often hidden in a polyclonal B-lymphocyte population. Immunoglobulin gene fingerprinting is a useful approach to screen for expanding clones and to detect recirculation between different locations. The gene fingerprinting approach and the Southern blot technique have been amalgamated, using electrophoretic transfer of a PCR product from an acrylamide gel onto a nylon membrane followed by hybridization with specific oligonucleotide probes. In contrast to conventional fingerprinting, the authenticity of immunoglobulin genes can be confirmed, individual genes can be detected and handling radionucleotides can be avoided. Also, the membrane may be reused for further investigations.
... On the basis of the clonal relationship between the B-CLL and HRS cell clones, this indicates that (a precursor of) the HRS tumor clone resided in the patient for several years before it gave rise to clinically apparent HD. Evidence that (premalignant?) precursors of the B-CLL tumor clone can indeed persist as expanded clones for long time besides the B-CLL, has been provided previously by Dono et al. (25). ...
Background: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. Materials and Methods: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. Results: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient I showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. Conclusions: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CDS-positive B cells.
