Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.

In the human, most IgM+IgD+ as well as CD5+ peripheral blood B cells express unmutated V genes and thus can be assigned to a pre-germinal centre (GC) stage of development. The memory B-cell compartment generated in the GC reaction and characterized by cells bearing somatically mutated V-region genes consists not only of class-switched cells, but also of IgM-only B cells and perhaps a subset of IgM+IgD+B cells expressing the CD27 antigen. Comparison of the rearranged V-region genes of human B-cell lymphomas with those of the normal B-cell subsets allows the identification of the progenitor cells of these tumours in terms of their stage of maturation. On this basis, most B-cell non-Hodgkin lymphomas, and in addition Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin's disease (HD), are derived from B cells at a GC or post-GC stage of development. The mutation pattern indicates that the precursors of the tumour clones have been stringently selected for expression of a functional antigen receptor with one notable exception: HRS cells in classical (but not lymphocyte-predominant) HD appear to be derived from "crippled" GC B cells. Sequence analysis of rearranged V genes amplified from single tonsillar GC B cells revealed that the somatic hypermutation process introduces deletions and/or insertions into V-region genes more frequently than indicated by previous investigations. Presumably, this feature of the hypermutation mechanism is often responsible for the generation of heavy chain disease, and also several types of chromosomal translocations of oncogenes into immunoglobulin loci in human B-cell lymphomas.

Human naive and germinal center (GC) B cells were sorted by f low cytometry and rearranged V H region genes were amplified and sequenced from single cells. Whereas no deletions or insertions were found in naive B cells, Ϸ4% of in-frame and >40% of out-of-frame rearrangements of GC B cells harbored deletions and͞or insertions of variable length. The pattern of deletions͞insertions and their restriction to mutated V genes strongly suggests that they result from somatic hypermutation. Deletions and insertions account for Ϸ6% of somatic mutations introduced into rearranged V H region genes of GC B cells. These deletions͞insertions seem to be the main cause for the generation of heavy chain disease proteins. Furthermore, it appears that several types of oncogene translocations (like c-myc translocations in Burkitt's lymphoma) occur as a byproduct of somatic hypermutation within the GC-and not during V(D)J recombination in the bone marrow as previously thought.During B cell differentiation in the bone marrow, somatic recombination of antibody gene segments is carried out by B cell progenitors to create functional heavy and light chain variable (V) region genes (1). Antibody diversity is generated by assembling one of the different V, (D), and J genes for each V region gene and by imprecise joining of these gene segments (2). Only B cells expressing functional heavy and light chain genes are allowed to enter the peripheral B cell pool as naive IgM ϩ IgD ϩ B cells (1). If peripheral B cells are activated by cognate antigen in the course of a T cell-dependent immune response, antibody V genes are further diversified by the introduction of somatic mutations in the microenvironment of the germinal center (GC) (3). The somatic mutations, which are introduced at a high rate (Ϸ10 Ϫ3 to 10 Ϫ4 ͞bp͞generation) to a region of Ϸ2 kb from the leader region to the J-C intron into rearranged heavy and light chain genes (4), mainly have been described as single nucleotide exchanges, whereas deletions͞insertions only rarely have been observed (4, 5).However, V genes harboring deletions and͞or insertions were detected repeatedly in our studies of rearranged V region genes in various human B cell lymphomas (6-12): Deletions and͞or insertions were found in 2 of 10 V H gene rearrangements and 1 of 14 V genes amplified from Burkitt's lymphomas (9), 1 of 4 V H genes from monocytoid B cell lymphomas (7), 1 of 14 V H and 1 of 11 V L genes from diffuse large cell lymphomas (8), 3 of 13 V H region genes from classical Hodgkin's disease (6, 10, 11), and 3 of 7 V H region genes from lymphocyte-predominant Hodgkin's disease (6, 12). A common feature of all of these lymphomas is that they carry somatically mutated V region genes and are thus derived from GC or post-GC B cells.The occurrence of deletions͞insertions within rearranged V genes has not been analyzed systematically in normal human B cells, so it is unclear whether their frequent occurrence in the lymphomas is a peculiar feature of malignant B cells or whether they are also present...

Southern blot analyses of immunoglobulin light chain gene rearrangements in human leukemias and myelomas indicated that λ loci in κ‐producing cells are largely unrearranged while κ loci in λ producers are often rearranged and inactivated by rearrangements of the kappa‐deleting element (KDE). For a systematic analysis of the regulation of light chain rearrangements during early B cell development in normal human B cells also considering functionality of the rearrangements, we used FACS‐sorted single naive κ‐ and λ‐expressing B cells from peripheral blood of healthy humans. VκJκ and VλJλ joints and rearrangements involving the KDE were amplified simultaneously from single cells and sequenced. Whereas only 2 – 3 % of κ‐expressing cells cary Vλ Jλ  joints, nearly all λ‐expressing cells have rearranged κ loci and indeed carry VκJκ joints. The VκJκ joints in λ‐expressing cells exhibit preferential Jκ4 and Jκ5 over Jκ1 and Jκ2 usage compared to κ‐expressing cells. Thirty percent of the VκJκ joints in λ producers are rearranged in‐frame. These data indicateextensive sequential Vκ‐Jκ rearrangements and inactivation of functional VκJκ joints in λ‐expressing cells, presumably before VλJλ joining.

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