Genetic Landscape of Ocular Adnexa Extranodal Marginal Zone Lymphoma

Background: Ocular adnexa lymphomas (OAL) consist of a heterogeneous group of tumors that can arise in the lacrimal gland, orbit, conjunctiva and eyelid. More than 95% of these lymphomas are of B-cell origin and extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue is the most common subtype of OAL, accounting for 55-85% of the cases. Ocular adnexa extranodal marginal zone lymphoma (OA-EMZL) patients have a persistent risk of relapses and in 4% of the cases, the disease transforms into an aggressive lymphoma. A comprehensive list of all somatic nonsilent mutations remains currently unknown thus hampering the complete understanding of the disease and the development of new diagnostic and therapeutic approaches. Previous studies analyzed only a small number of OA-EMZL patients using gene targeted sequencing. In this study we utilized whole-exome sequencing to define the genetic landscape of 69 cases of OA-EMZL. Our data provide an unbiased identification of genetically altered genes and pathways that may play a role in the molecular pathogenesis of OA-EMZL.

Methods: GATK HaplotypeCaller was used for joint variant calling on all samples, while MuTect was used to detect somatic variants in seven tumor samples with available paired normal germline DNA. We focused on variants that were exonic, not synonymous, and deleterious (CADD score > 10), while excluding any variants found with a frequency > 1% in control populations. For copy number (CN) analysis we used EXCAVATOR on each sequenced exome in pooled mode against the seven paired normal samples. The output was provided to GISTIC 2.0 to determine recurrent arm-level and gene-level CN changes.

Results: We identified 125 candidate cancer driver genes with the following characteristics: 1) have genomic alteration in more than 5% of the cases, 2) are present in the list of COSMIC and DLBCL driver genes, 3) are considered essential genes or tumor suppressor genes as previously defined in a functional CRISPR screen (Reddy et al., Cell 2017). Among the most frequent alterations there were mutations and CN losses of CABIN1 (32%), inactivation of TNFAIP3 (26%) and KMT2D (22%), mutations and CN changes in CARD11 (25%), RHOA (25%), CREBBP (20%) and TBL1XR1 (23%). Gene set enrichment analysis of candidate driver genes showed statistically significant enrichment for genes involved in cell junction, adhesion, cytoskeleton regulation, chromatin organization and for genes harboring at least one highly conserved NFAT binding motif TGGAAA in their promoter. In addition, we identified recurrent lesions affecting several genes belonging to the NFkB, NOTCH and B cell receptor (BCR) signaling pathways. The most frequent alterations in the NFkB pathway included inactivation of TNFAIP3 (26%), mutations and CN alterations of CARD11 (25%), MYD88 (12%), BCL10 (10%) and CRD10 (9%). Mutations affecting NOTCH signaling were found in NOTCH3 (22%), DTX1 (12%), MAML1 (12%), MAML2 (12%), DLK1 (10%) and SPEN (9%). BCR signaling genes alterations were observed in PIK3R2 (12%), VAV1 (10%), PTPN6 (7%) and BTK (4%).

CABIN1 was one of the most commonly altered genes. In T cells, CABIN1 is implicated in inhibiting calcineurin-mediated signal transduction, including NFAT activation. In reactive lymph node and tonsil tissue, CABIN1 was expressed in reactive B cells (CD20⁺ and CD3^-) localized mainly in the mantel zone area, as demonstrated by IHC and IF. We hypothesized that it may act as a negative regulator of BCR signaling in MZL. To verify this possibility we used shRNAs to knock down CABIN1 in MZL cell lines and measured NFAT activity in response to IgM stimulation using a luciferase reporter system. IgM stimulation of CABIN1-depleted MZL cells induced a more pronounced activation of NFAT signaling compared to wild-type cells.

Conclusions: The genetics of OA-EMZL is characterized by somatic alterations in NFkB, NOTCH, and BCR signaling pathways. Candidate cancer driver genes are implicated in chromatin organization, cytoskeleton regulation and in adhesion, and they are also enriched for NFAT target genes. One of the most altered candidate cancer driver genes was CABIN1, which is a calcineurin inhibitor that acts as a negative regulator of NFAT activity. CABIN1 depletion in MZL cells boosts BCR-stimulated NFAT activity. Our data suggest a widespread dysregulation of NFAT signaling cascade downstream of BCR stimulation as a possible mechanism of OA-EMZL pathogenesis.