RESUMO
Normal IgM(-)IgD(+) CD38(+) B cells and IgM(-)IgD(+) multiple myelomas (MM) are characterized by Cmu deletion, biased Iglambda expression and hypermutated IgV regions. The predominant Iglambda usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal expansion in the germinal center environment. Here, four cases of IgDlambda MM were studied to address the question of light chain receptor revision in a 'single cell' model. Detailed analyses of both IGK and IGL alleles of each case were performed by Southern blotting, (RT-) PCR, and sequencing. The expressed IgV genes were extensively mutated and Cmu deletion was confirmed in two cases. In addition, in the four MM a total of six non-functional deletional IGK rearrangements were identified, which proved to be unmutated. We conclude that IgD myelomas indeed originate from (post) germinal center B cells in which, in spite of the fact that they are hypermutated, there is no evidence of receptor revision.
Assuntos
Imunoglobulina D/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Mieloma Múltiplo/genética , Linfócitos B/imunologia , Linfócitos B/patologia , Sequência de Bases , Rearranjo Gênico de Cadeia Leve de Linfócito B , Humanos , Cadeias lambda de Imunoglobulina/biossíntese , Dados de Sequência Molecular , Mieloma Múltiplo/imunologia , Mutação , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
In this study we describe alternative splicing of somatically mutated immunoglobulin (Ig) variable heavy chain (V(H)) genes in three distinct primary B cell non-Hodgkin's lymphomas (B-NHL). In two V4-34 expressing lymphomas, ie a post-germinal center type B cell chronic lymphocytic leukemia (B-CLL) and a follicular lymphoma (FL), internally spliced V(H) gene transcripts were found in which a sequence stretch of 116 bp between the framework region 1 (FR1) and complementarity determining region 2 (CDR2) had been deleted. We provide evidence that for this alternative IgV(H) mRNA processing a known cryptic 5' splice donor site and a previously unidentified cryptic 3' splice acceptor site were used. Site-directed mutagenesis showed that the cryptic 3' splice acceptor site had been activated by specific somatic point mutations. The B-CLL further harbored a triplication of the rearranged JH3 gene segment including the putative N region and part of the JH3-JH4 intron sequence. This triplication probably took place via a repeated mechanism of DNA double strand break followed by homologous recombination, a mechanism which was recently proposed also involved in the somatic hypermutation process and is compatible with the post-germinal center derivation of this B-CLL. Finally, in a V4-34 expressing diffuse large B cell lymphoma, we observed alternative IgV(H) mRNA processing using the same cryptic 5' splice donor site and the normal splice acceptor site of the CH1-C(mu) exon. The significance of alternative IgV(H) processing in B cell malignancies and as a potential mechanism of somatic Ig diversification is discussed.
Assuntos
Processamento Alternativo/genética , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Idoso , Sequência de Bases , Regiões Determinantes de Complementaridade/genética , Sequência Consenso , Primers do DNA/química , DNA de Neoplasias/metabolismo , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Neoplásico/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
To investigate B-cell receptor evolution in follicular lymphomas (FLs), immunoglobulin variable heavy chain (V(H)) gene regions of 3 FLs were analyzed at different time points. One FL with a high somatic mutation load and intraclonal V(H) gene diversity was investigated in situ. V(H) gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells isolated from frozen tissue sections by laser microdissection. Interestingly, the mutation pattern of the prevalent subclone in the relapse biopsy was virtually identical to that of a subclone isolated by microdissection from the presentation biopsy 9 years earlier. In a second FL, proof was obtained that the subclone that dominated the relapse sample had already been present in the initial biopsy. The finding that subclones found in the relapses of these FLs had not evolved over time but were preexistent, challenges the concept of antigen-driven B-cell receptor evolution during disease course.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Linfoma Folicular/genética , Linfoma Folicular/imunologia , Transformação Celular Neoplásica , Células Clonais , Secções Congeladas , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Região Variável de Imunoglobulina , Linfonodos/imunologia , Linfonodos/patologia , Linfoma Folicular/patologia , Receptores de Antígenos de Linfócitos B/genética , Fatores de TempoRESUMO
The expansion of follicular lymphomas (FLs) resembles, both morphologically and functionally, normal germinal center B-cell growth. The tumor cells proliferate in networks of follicular dendritic cells and are believed to be capable of somatic hypermutation and isotype switching. To investigate the relation between somatic mutation and heavy chain isotype expression, we analyzed the variable heavy (V(H)) chain genes of 30 FL samples of different isotypes. The V(H) genes of the FLs were heavily mutated (29.3 mutations on average). In addition, isotype-switched lymphomas contained more somatic mutations than immunoglobulin M-positive lymphomas (33.8 mutations per V(H) gene versus 23.0, respectively). In all but one of the FLs, the ratios of replacement versus silent mutations in the framework regions were low, independent of the absolute number of somatic mutations and the level of intraclonal variation. Analysis of relapse samples of 4 FLs showed no obvious increase in somatic mutation load in most FLs and a decrease in intraclonal variation in time. In 3 of 4 cases, we obtained evidence for selection of certain subclones, rather than clonal evolution. Our findings question if intraclonal variation is always a reflection of ongoing somatic hypermutation. This may have implications for the concept of antigen-driven lymphomagenesis. (Blood. 2000;95:2922-2929)
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfoma Folicular/genética , Linfoma Folicular/imunologia , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Clonagem Molecular , Genes de Imunoglobulinas , Variação Genética , Humanos , Imunoglobulina G/genética , Isotipos de Imunoglobulinas/genética , Imunoglobulina M/análise , Imunoglobulina M/genética , Região de Troca de Imunoglobulinas , Linfonodos/imunologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Alinhamento de SequênciaRESUMO
Primary cutaneous B-cell lymphomas are B-cell non-Hodgkin's lymphomas that arise in the skin. The major subtypes discerned are follicle center cell lymphomas, immunocytomas (marginal zone B-cell lymphomas), and large B-cell lymphomas of the leg. In this study, we analyzed the variable heavy chain (VH) genes of 7 of these lymphomas, ie, 4 follicle center cell lymphomas (diffuse large-cell lymphomas) and 3 immunocytomas. We show that all these lymphomas carry heavily mutated VH genes, with no obvious bias in VH gene usage. The low ratios of replacement versus silent mutations observed in the framework regions of 5 of the 7 lymphomas suggest that the structure of the B-cell antigen receptor was preserved, as in normal B cells that are selected for antibody expression. Moreover, evidence for ongoing mutation was obtained in 3 immunocytomas and in one lymphoma of large-cell type. In addition, in 1 immunocytoma, both IgG- and IgA-expressing clones were found, indicative of isotype switching. Our data provide insight into the biology of primary cutaneous B-cell lymphomas and may be of significance for their classification.
