V(D)J recombination and staggered DNA breaks: guilty again

In this issue of Blood, Murga Penas and colleagues describe mechanisms involved in the genesis of the t(14;18)(q32;q21)/IgH-MALT1 in MALT lymphoma, and they identify a cluster region suitable for detection by polymerase chain reaction.

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue, also known as MALT lymphoma, arises in extranodal sites. The most common sites are stomach, lung, skin, ocular adnexae, salivary glands, and thyroid gland, but virtually any extranodal site can be involved.¹  MALT lymphoma is a prototype for antigen-driven B-cell lymphomagenesis. Antigens involved in lymphomagenesis result from either chronic infection (eg, Helicobacter pylori in stomach) or autoimmune diseases (eg, Hashimoto thyroiditis, Sjogren syndrome). Most extranodal sites involved by MALT lymphoma normally lack lymphoid tissue. Chronic antigenic stimulation results in these sites acquiring lymphoid tissue, within which MALT lymphomas can arise.

Chromosomal translocations, usually resulting in oncogene activation, occur in many types of lymphoma, and their detection is helpful for establishing a diagnosis and monitoring disease after therapy. In MALT lymphomas, a total of 10 chromosomal translocations have been reported, of which 4 are well characterized and implicated in pathogenesis, with 6 more recently described and variably defined.^1,2  t(11;18)(q21;q21) is most common, identified in up to one-third of cases, and results in a chimeric API2-MALT1 gene. t(14,18)(q32;q21)/IgH-MALT1, t(3,14)(p14.1;q32)/IgH-FOXP1, and t(1,14)(p22;q32)/IgH-BCL10 have been identified in approximately 10% to 20%, 10%, and 1% to 2% of MALT lymphomas, respectively, and result in overexpression of MALT1, FOXP1, and BCL10. These translocations are mutually exclusive in an individual MALT lymphoma and correlate with site of disease.³  MALT lymphomas associated with t(11;18) arise most often in the stomach and lung. In contrast, MALT lymphomas associated with t(14;18) arise commonly in skin, ocular adnexae, salivary glands, and liver; MALT lymphomas associated with t(3;14) tend to occur in the thyroid gland, skin, and the ocular adnexae; and MALT lymphomas associated with t(1;14) may have a predilection for the intestines. The heterogeneity of MALT lymphomas, in their site of origin, associated diseases, and translocations, suggests that MALT lymphoma is actually a number of different, but related, diseases.

The immunoglobulin heavy chain gene (IgH) is composed of a number of variable (V), diversity (D), joining (J), and constant region genes. In normal B cells these genes recombine via formation of double-stranded DNA breaks to generate functional Ig genes and protein with high antigen affinity. This process, known as V(D)J recombination, is the main mechanism for generating antigen-receptor diversity. The breaks and recombination events that occur during this process explain the tendency for chromosomal translocations in B-cell lymphomas to involve the Ig loci. However, the precise molecular mechanisms underlying these translocations remain unknown and different mechanisms have been proposed in various lymphoma types.

In this issue of Blood, Murga Penas and colleagues show that the t(14;18)(q32;q21) is the result, at least in part, of illegitimate V(D)J recombination.⁴  By analyzing the sequence of the breakpoints at the IgH locus on chromosome 14, they identified findings typical of V(D)J-mediated recombination, that is, presence of a recombination signal sequence and evidence of coding end processing, including nucleotide deletions and insertion of non–template-dependent (N) nucleotides. The authors also found insertions of templated (T)–nucleotides at the junction sites. T-nucleotides are short copies of at least 5 nucleotides copied from the regions surrounding the breakpoints and often contain point mutations and/or insertions or deletions. Because T-nucleotides have not been described in normal V(D)J recombination products, their presence suggests that they are generated by error-prone template-dependent DNA synthesis rather than illegitimate V(D)J recombination. In contrast, analysis of the chromosome 18 breakpoints showed findings inconsistent with V(D)J recombination mechanisms. The authors identified an 87 base pair (bp) region in which the breakpoints of all cases clustered in the 5′ noncoding region of MALT1. In at least one case, analysis of the DH-MALT1 junction showed a duplication of 8 MALT1 nucleotides, suggesting a staggered double-stranded DNA break. Similar findings have been found in other chromosomal translocations, such as t(14;18)/IgH-BCL2 in follicular lymphoma and t(11;14)/CCND1-IgH in mantle cell lymphoma, suggesting that these chromosomal translocations are generated by similar mechanisms.^5,6  In these translocations, illegitimate V(D)J mechanisms mediate recombination at the IgH locus, T-nucleotides are reported at the breakpoints, and breakpoint clusters occur in the BCL2 and CCND1 loci.

Another interesting implication of this study is the timing of translocations: when in B-cell differentiation do they occur? It seems intuitive that a particular chromosomal translocation may arise at a specific stage of B-cell differentiation, and therefore contributes to the biologic and clinical features of the neoplasm. In the case of MALT lymphomas, it would be expected that chromosomal translocations arise after the B cell has encountered antigen within the primary site of disease (eg, stomach). In the cases analyzed, the locations of the few somatic mutations identified suggest that they most likely resulted from error-prone repair of end-joining, rather than from the process of somatic mutation. These cases suggest, therefore, that MALT lymphomas arise from a B cell that encountered antigen outside the context of the germinal center microenvironment.

The diagnosis of MALT lymphoma can be challenging for pathologists. The problems in the diagnosis are related in part to the small size of biopsy specimens and the lack of specific immunophenotypic markers useful for diagnosis. For this reason, the identification of a novel, 87-bp cluster region in the t(14,18)(q32;q21)/IgH-MALT1 will facilitate the development of polymerase chain reaction assays useful for the diagnosis in both fresh and paraffin-embedded biopsy specimens and will be helpful for the evaluation of minimal residual disease.