Marginal zone lymphoma (MZL) is a B-cell non-Hodgkin lymphoma (B-NHL) derived from marginal-zone B-cells. The current WHO classification recognizes three types of MZLs, which comprise extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Four recurrent and mutually exclusive chromosomal translocations: t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), and t(3;14)(p14.1;q32) have been described in EMZLs, but not in NMZLs or SMZLs, with frequencies ranging from 0–40% depending on differing anatomic sites and geographic regions. At least three of these translocations, t(11;18) API2/MALT1, t(1;14) IgH/BCL10, and t(14;18) IgH/MALT, result in constitutive activation of nuclear factor-kB (NF-kB), a transcription factor complex regulating multiple cellular processes including cell growth and survival. Approximately 25% of MZL however, lack any recognizable recurrent genetic alteration. Recently, biallelic deletions of the chromosomal region 6q23.3-q24.1 containing the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), which is a negative regulator of NF-kB, were detected in ocular adnexal MZLs, suggesting a role for A20 as a tumor suppressor in this disease entity. Here, we investigated a panel of 32 cases representing all three types of MZLs by PCR amplification and sequencing of the A20 coding exons. FISH was also performed using locus specific probes for A20 and Blimp1. Five 1–2 bp deletions and a single bp insertion, all leading to premature stop codons, were identified in 5/20 (25%) non-splenic MZLs and 1/12 (8.3%) SMZLs. The somatic nature of the mutations was confirmed by analyzing matched normal DNA in 4 cases. All A20 mutations were predictive of severely truncated polypeptides lacking functionally relevant domains. Genetic loss of A20 was detected by FISH in 4/20 (20%) non-splenic MZLs, including 1 with biallelic loss, but not in SMZLs. Two of the four A20 deletions seen by FISH were also observed by array-CGH. Deletions encompassing Blimp1 were not detected in cases with A20 deletions. No evidence for epigenetic silencing of A20 via promoter methylation of CpG islands was found in 10 MZLs analyzed. Thus, biallelic inactivation of A20, either via deletion of both alleles or frameshift mutations of one allele and loss of the second allele was identified in 3/20 (15%) non-splenic MZLs, while an additional 4/20 (20%) non-splenic MZLs displayed monoallelic A20 inactivation by mutations (N=3) or deletion (N=1). When we determined the relationship between A20 inactivation and trisomy 18 or the translocations associated with constitutive activation of NF-kB, A20 inactivation appeared mutually exclusive with these cytogenetic aberrations, with the exception of one case that had trisomy 18. In summary, our results indicate that A20 is mono- or biallelically inactivated in approximately one third of MZLs, arising in both nodal (3/9, 33.3%) and extranodal sites (4/11, 36.4%), and in a minority of SMZLs (1/12, 8.3%). Evidence of a classic two-hit mechanism was observed in 3/20 (15%) non-splenic MZL. In addition, detection of MZLs with monoallelic inactivation suggests a haploinsufficient role of A20 as a tumor suppressor. Thus, A20 inactivation may represent a common mechanism for constitutive activation of the NF-kB pathway, which, in turn, may contribute to lymphomagenesis by stimulating cell proliferation and survival. The identification of genetic lesions in MZLs that deregulate NF-kB activity offers the possibility of targeted therapeutic intervention.

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