TNFAIP3 (A20) Genetic Alterations In EBV Associated AIDS Related Lymphomas

AIDS related lymphomas (ARL) are a heterogeneous group of lymphoproliferative disorders that are frequently associated with Epstein Barr virus (EBV) infection. EBV expresses latent viral oncoproteins that constitutively activate the transcription factor NF-κB, a potent inducer of genes involved in B cell survival and proliferation (Keller SA et al, Blood 2006). Lymphomas that are not associated with EBV can also display increased NF-κB activity and recent reports have described mutations in regulators of NF-κB in subsets of B cell lymphomas. One of the frequently mutated regulatory genes is TNFAIP3, which encodes A20, an ubiquitin modifying enzyme involved in the termination of NF-κB signaling. Mutations resulting in the inactivation of A20 have been found in a significant proportion of marginal zone lymphoma (Novak U et al, Blood 2009), classical Hodgkin lymphoma, primary mediastinal B cell lymphoma (Schmitz R et al, J Exp Med 2009), and diffuse large B cell lymphoma (Compagno M et al, Nature 2009). In ARL the incidence of alterations in A20 and the relationship with EBV infection has not been described.

We evaluated archival formalin fixed paraffin embedded tissue samples of ARL for genetic alterations in A20. Tissue was collected through an international collaboration between Weill Cornell Medical College in New York, USA and Siena University in Siena, Italy. A tissue microarray with 46 cases of ARL was prepared and characterization of lymphoma subtype and EBV viral status were determined by immunohistochemistry and in situ hybridization for Epstein-Barr encoded RNA. Fluorescent in situ hybridization (FISH) was used to evaluate for genomic deletions in A20, and translocations of cMYC, BCL-2 and BCL-6. Direct sequencing of the coding region and splice sites of A20 was performed to evaluate for additional genetic alterations. Immunohistochemistry was used to evaluate for the presence of A20 protein.

Fluorescent in situ hybridization revealed A20 monoallelic or biallelic deletion in 6 of 25 cases (24%). A20 point mutations were found in 3 of 23 cases (13%). Nonsense mutations coding for a premature stop codon in exon 2 were seen in 2 cases. The third case was found to have a missense mutation in exon 7 resulting in an amino acid change. Two of the 3 cases with an A20 point mutation had A20 deletion in the complementary allele indicating biallelic alteration of the A20 gene. Immunohistochemistry for A20 was performed and is reported for the first time in this abstract. Absence of A20 protein was demonstrated in 4 of 33 samples (12%). Included among the cases negative for A20 on immunohistochemistry is the single case with biallelic A20 deletion demonstrated by FISH. In total 10 of 39 (26%) cases with adequate sample for evaluation were determined to have inactivation of A20 by FISH, sequencing, immunohistochemistry, or a combination. A20 inactivation was seen among all histologic subtypes of ARL including Burkitt lymphoma (n=2), diffuse large B cell lymphoma of the germinal center B cell (n=2) and non-germinal center B cell (n=2), plasmablastic lymphoma (n=3) and B cell lymphoma, unclassifiable, intermediate between BL and diffuse large B cell lymphoma (n=1). Interestingly, the incidence of EBV infection was higher in cases with A20 inactivation than in those with intact A20. EBV was present in 6/10 cases with A20 alteration (60%) vs. 8/29 cases with intact A20 (28%). The EBV latent viral protein LMP-1, which activates NF-κB, was not expressed in cases with A20 alteration.

This is the first report to demonstrate A20 inactivation in EBV-associated lymphoma. A20 molecular analysis has been previously reported in Hodgkin Lymphoma (HL) where A20 inactivation and EBV infection were found to be almost mutually exclusive (Schmitz R et al, J Exp Med 2009). The EBV gene expression pattern differs in HL and ARL. In HL EBV expresses the viral oncoprotein LMP-1, which leads to constitutive activation of NF-κB. In ARL viral gene expression is more heterogeneous and in this cohort of ARL, LMP-1 was not expressed in any of the cases with EBV infection and A20 loss. Our data indicate that A20 may represent a tumor suppressor gene in a significant subset of ARL and that A20 inactivation may be associated with positive EBV status. In EBV related lymphoma inactivation of A20 may be an alternative mechanism of NF-κB upregulation in the absence of LMP-1.

No relevant conflicts of interest to declare.