Wilhelmina M. Aarts

Primary Follicular Lymphoma of the Small Intestine: α4β7 Expression and Immunoglobulin Configuration Suggest an Origin from Local Antigen-Experienced B Cells

Primary follicular lymphoma of the gastrointestinal tract (GI-FL) is a rare so far poorly studied entity. We analyzed four FL cases located in the small intestine and duodenum to gain insight in their pathogenesis and to find an explanation for their low tendency to disseminate outside the GI tract. GI-FLs resemble nodal FLs with respect to morphology and expression of typical GC markers such as CD10, CD38, and BCL-6. We established that the high levels of the anti-apoptosis protein BCL-2 in the tumor cells are in all cases due to a t(14;18) involving the immunoglobulin heavy chain and BCL-2 loci. Detailed immunoglobulin gene analyses on microdissected tissue samples further supported the GC-cell derivation: GI-FLs carry extensively mutated variable heavy-chain genes. The mutation patterns indicated that at some time point in development stringent antigen receptor-based selection processes must have occurred. Interestingly, three of four neoplasms expressed surface IgA, an immunoglobulin class typical of the mucosal immune system and seldom found in nodal FL. In contrast to nodal FLs, the GI-FLs expressed the alpha4beta7 integrin, an established mucosa-homing receptor also expressed by normal intestinal B and T lymphocytes and by low-grade mucosa-associated lymphoid tissue lymphomas. However, the chemokine receptor CXCR3, expressed on low-grade mucosa-associated lymphoid tissue lymphomas, was not detected on the GI-FLs or on nodal FLs. The combined data suggests that primary FL of the small intestine is a distinct entity that originates from local antigen-responsive B cells.

In situ analysis of the variable heavy chain gene of an IgM/IgG-expressing follicular lymphoma - Evidence for interfollicular trafficking of tumor cells

It is generally assumed that follicular lymphomas (FL) not only morphologically resemble normal germinal centers but have retained some functional characteristics of their non-neoplastic counterparts as well. Recent IgV gene analyses on a panel of FLs however, strongly suggested that FLs do not retain the capacity of somatic hypermutation and are not being selected on basis of the quality of their mIgV regions. To extend these findings, we investigated the follicular organization and class switching in a FL that consisted of both IgM- and IgG-expressing tumor cells with a high somatic mutation load and significant intraclonal V(H) gene diversity. V(H)-C(mu) and V(H)-Cgamma gene transcripts were amplified and sequenced from samples of approximately 50 tumor cells, isolated from frozen tissue sections by laser microdissection. We identified many different subclones and obtained limited evidence of subclone dominance in individual follicles. Remarkably, several subclones were found scattered over different follicles. All samples contained IgM- and IgG-expressing tumor cells with, in general, non-identical mutation patterns, which is not in support of ongoing class switching. Accordingly, no switch circle recombination products were found. The findings indicate that the neoplastic follicles lack the organization and functions typical of reactive germinal centers.

Biased Iglambda expression in hypermutated IgD multiple myelomas does not result from receptor revision.

Normal IgM−IgD+ CD38+ B cells and IgM−IgD+ multiple myelomas (MM) are characterized by Cμ deletion, biased Igλ expression and hypermutated IgV regions. The predominant Igλ usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal expansion in the germinal center environment. Here, four cases of IgDλ 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 Cμ 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.

Immunoglobulin diversification in B cell malignancies: internal splicing of heavy chain variable region as a by-product of somatic hypermutation

In this study we describe alternative splicing of somatically mutated immunoglobulin (Ig) variable heavy chain (VH) genes in three distinct primary B cell non-Hodgkins 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 VH 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 IgVH 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 IgVH mRNA processing using the same cryptic 5′ splice donor site and the normal splice acceptor site of the CH1-Cμ exon. The significance of alternative IgVH processing in B cell malignancies and as a potential mechanism of somatic Ig diversification is discussed.

Analysis of Variable Heavy and Light Chain Genes in Follicular Lymphomas of Different Heavy Chain Isotype

Follicular lymphomas (FLs) are considered prototypes of germinal center derived B-cell malignancies. This contention is based on histomorphological characteristics [1] as well as on the presence of somatic mutations in the variable heavy and light chain genes (VH and VL genes) [2,3]. FLs are believed to have retained two important features of germinal center B cells, i. e. their capability of ongoing somatic hypermutation [4–6] and heavy chain isotype switching [7–9]. To investigate somatic mutation in relation to heavy chain isotype expression, we analyzed the immunoglobulin variable genes (IgV genes) of a panel of IgM- expressing and isotype-switched FLs. We find elevated frequencies of somatic mutation compared to normal B cells, but FLs resemble normal B cells in VH family gene usage, patterns of somatic mutation and differences in mutation frequency found between IgM-expressing and isotype-switched FLs. Our study questions the capacity of at least some FLs to undergo somatic hypermutation.