1,720,981 research outputs found
Defining origins and behaviour of malignant B-cells by immunoglobulin IgH locus analysis
No full textCD27 in defining memory B-cell origins in Waldenstrom’s macroglobulinemia
No full textCD27 is a tumor necrosis factor receptor family glycoprotein, identified in seminal studies as an apparently robust marker for normal memory B cells. Somatic hypermutation (SHM) in immunoglobulin variable (V) region genes, however, remains the definitive memory imprint. In Waldenström's macroglobulinemia (WM), SHM defines a predominant mutated (MUT) subset and a minor unmutated subset indicative of naive B-cell origin. In MUT-WM, tumor cells can lack CD27 expression, raising suggestions of unusual memory B-cell origins. We recently identified such normal IgM+D+CD27-ve memory B-cells, with low levels of SHM in VH genes. While these could seed WM, the possibility remains that WM could derive from classical memory B cells that shed CD27. The utility of CD27 expression in defining memory in MUT-WM origins, then, is uncertain, but SHM unequivocally defines memory B-cell derivation in most WM. Patterns of SHM and additional IgH locus events furthermore reveal ongoing intra-tumoral diversification in W
Determining mutational status of immunoglobulin v genes in chronic lymphocytic leukemia: a useful prognostic indicator
No full textDetermining the clonal origins of malignant B-cells will have an impact on disease understanding and management. In this regard, immunoglobulin variable (V) region gene analysis already is having a significant impact in delineating the tumor cell of origin. It can identify, among other features whether such a cell has undergone somatic mutation, which usually occurs within germinal centres. Remarkably, in chronic lymphocytic leukemia (CLL), the mutational status of V genes has allowed researchers to identify two subsets of disease, one originating from an unmutated B-cell with a markedly poorer disease outcome and the other from a mutated B-cell, which associates with long-term survival. The V gene status in CLL thus provides a robust indicator of disease outcome, which is beginning to shape clinical treatment. This chapter describes in detail the methodology for determining V gene usage in CLL, from acquisition of patient sample to generating the V-gene readout
Mantle cell lymphoma with t(11;14) and unmutated or mutated VH genes expresses AID and undergoes isotype switch events
No full textIsotype switch commonly follows onset of somatic hypermutation in the germinal center (GC), with activation-induced cytidine deaminase (AID) as a prerequisite. Mantle cell lymphoma (MCL) with t(11;14) includes a subset with unmutated (UM) and a minor subset with mutated (MUT) VH genes. Here, we investigated whether switch events and AID expression occur in MCL. In 4 of 6 UM and 4 of 7 MUT MCLs, alternative tumor-derived C?,?,? transcripts were identified. AID transcripts, including a splice variant, were common to both subsets. AID expression correlated with switch in 8 of 8 cases, but in 3 of 5 cases it occurred with switch absent. Circle transcripts (I?-Cµ/I?-Cµ) were identified in 5 of 7 evaluated cases. In 1 of 12 cases, 12% of tumor cells expressed immunoglobulin L-restricted surface IgA. Ongoing switch recombination events appear to be a feature of MCL, likely restricted to a minor tumor subpopulation, with occasional variant sIg expression. UM MCLs implicate origins from pre-GC B cells and reveal switch events at ectopic sites. (Blood. 2004;103:2795-2798
Dynamics of activation induced cytidine deaminase expression in t(11;14) mantle cell lymphoma
No full textIgM-expressing Waldenstrom's Macroglobulinemia tumor cells reveal a potential for isotype switch
No full textIn Waldenstrom’s macroglobulinemia (WM), which locates primarily in the bone marrow (BM), VH gene analysis had previously suggested origins from a post-follicular B-cell arresting prior to isotype switch. Using more sensitive assays, facilitated by amplified cDNA from BM cells, nested PCR unexpectedly revealed tumor-derived isotype-switch transcripts in 7/7 cases. In 5/7 cases, both C and C variant transcripts were identified, and C or C only in 2/7. Detection of activation induced cytidine deaminase (AID) and germline and circle transcripts confirmed switching activity. Selected gene expression profiles established the memory B-cell marker CD27 as highly expressed in all cases. These findings were evaluated further in additional WM cases where availability of tumor material allowed detailed analysis. In 2/2 cases, phenotype suggested a variable CD27 expression within the tumor clone. In these, tumor IgM transcripts were readily detected in both the CD19+CD27+ and CD19+CD27– fractions, and in 1 of the 2 cases, post-switched tumor-derived C transcripts were also identified in each fraction. In this WM case, the frequency of tumor-derived transcripts was then assessed at the single cell level. Switch transcripts were identified in 3/96 cells with no co-expression of the IgM isotype. Similarly, AID transcripts were observed in some cells, not always correlating with switch events or with ongoing somatic mutation, which was apparent in VDJ-Cµ sequences. These findings reveal a dynamic intratumoral diversification, with AID activated and ongoing mutational and switch activity occurring post-transformation in a proportion of the tumor clone. Heterogeneity in CD27 expression is also evident within tumor cells, revealing phenotypic change. Interestingly, these data indicate that although WM tumor cells have arrested at the IgM stage and do not express isotype switched Ig, they retain the capacity to initiate events critical for isotype switch
High-affinity memory B cells induced by conjugate vaccines against weak tumor antigens are vulnerable to nonconjugated antigen
No full textInduction of antibody-mediated immunity against hematologic malignancies requires CD4+ T-cell help, but weak tumor antigens generally fail to induce adequate T-cell responses, or to overcome tolerance. Conjugate vaccines can harness alternative help to activate responses, but memory B cells may then be exposed to leaking tumor-derived antigen without CD4+ T-cell support. We showed previously using lymphoma-derived idiotypic antigen that exposure to “helpless” antigen silences the majority of memory IgG+ B cells. Transfer experiments now indicate that silencing is permanent. In marked contrast to IgG, most coexisting IgM+ memory B cells exposed to “helpless” antigen survive. Confirmation in a hapten (NP) model allowed measurement of affinity, revealing this, rather than isotype, as the determinant of survival. IgM+ B cells had Ig variable region gene usage similar to IgG but with fewer somatic mutations. Survival of memory B cells appears variably controlled by affinity for antigen, allowing a minority of low affinity IgG+, but most IgM+, memory B cells to escape deletion in the absence of T-cell help. The latter remain, but the majority fail to undergo isotype switch. These findings could apply to other tumor antigens and are relevant for vaccination strategies aimed to induce long-term antibody
Immunoglobulin heavy chain locus events and expression of activation-induced cytidine deaminase in epithelial breast cancer cell lines
Full text linkWhen cells transform, phenotypic and genetic profiles can be dramatically altered. Nevertheless, a recent report identifying IgG in breast cancer cells was unexpected, revealing differentiation features normally associated with B lymphocytes. To extend these findings, we focused on immunoglobulin variable (V) region gene analysis using well-defined breast cancer cell lines expressing the epithelial marker, epithelial cell adhesion molecule (EpCAM). VH gene transcripts were identifiable by nested reverse transcription-PCR either as single or dual V, diversity (D), and joining (J) rearrangements in four of six lines, most being potentially functional. V(D)J transcripts were observed in sequential cultures, indicating stable expression. To exclude coexisting lymphocytes, each cell line was shown to be EBV negative, with CD19/CD20 and cytoplasmic/surface immunoglobulin also absent by flow cytometry. Identified VH transcripts were then sought in individual tumor cells, isolated as EpCAM+ single cells by flow cytometry. Importantly, in three of three selected cell lines, VH genes were identifiable in a significant fraction (~32%) of single cells. In five of six identified VH genes, somatic mutations were apparent with no intraclonal variation, indicating cessation of mutational activity. VH transcripts were pre- and post-isotype switch, with activation of switch events evident from expressed germ-line switch transcripts in two of six lines. Strikingly, six of six cell lines expressed activation-induced cytidine deaminase (AID) essential for mutational and switch activity. These data suggest either a de novo rearrangement and modification of VH genes in epithelial tumor cells or assimilation of lymphocyte-derived chromatin. Constitutive AID activation in malignant epithelial cells further raises a potential for inducing aberrant mutational activity
Hairy cell leukemia: at the crossroad of somatic mutation and isotype switch
No full textHairy cell leukemia (HCL) commonly expresses multiple immunoglobulin isotypes, a feature rare in other B-cell malignancies or in normal B cells. In HCL, there is no phenotypic evidence for subpopulations, and single cells from one previous case contained transcripts for several isotypes. This raises the questions of the differentiation status of the cell of origin and of posttransformation events. We have investigated 9 cases, all expressing multiple immunoglobulin isotypes. Multiple tumor-derived variable-(diversity)-joining-constant ?, ?, ? (V(D)J-Cµ ?, ?, ?) transcripts were confirmed in single cells of a further case. All cases were negative for germinal center (GC)-associated markers CD27 and CD38. Seven of 9 cases had mutated VH genes, with low levels of intraclonal heterogeneity, but 2 of 9 were unmutated, indicative of pre-GC origin. Eight of 9 cases expressed activation-induced cytidine deaminase (AID), a molecule essential for somatic mutation and isotype switch. All cases expressed germ line heavy-chain I exon (IH)-CH transcripts which paralleled surface immunoglobulin (sIg) isotype. Significantly, no circle transcripts indicative of deletional recombination of switched isotypes were detectable in 9 of 9 cases. These data indicate heterogeneity in the cell of origin in terms of mutational status, but reveal common features of AID expression and isotype-switching events occurring prior to deletional recombination. Both mutational and switching events may be influenced by environmental factors at extrafollicular sites
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