MicroRNA Profiling in Low-Grade Ocular MALT and Diffuse Large B-Cell Lymphoma: A Role for MYC and NFKB1 Mediated Dysregulation of MicroRNA Expression in Aggressive Disease
Authors: Hother Christoffer , Rasmussen Peter Kristian , Joshi Tejal , Reker Ditte , Ralfkiaer Ulrik , Workman Christopher , De Nully Brown Peter , Heegaard Steffen , Ralfkiaer Elisabeth , Grønbæk Kirsten 
Published: October 2019

Abstract
Abstract 1600

Introduction:
Although rare, ocular adnexal lymphomas (i.e. lymphoma of the orbit, eyelids, conjunctiva, lacrimal gland and lacrimal sac), are among the most common malignant tumors involving the ocular adnexal regions. The main subtypes are low-grade mucosa associated lymphoid tissue (MALT) lymphoma and aggressive diffuse large B cell lymphoma (DLBCL). In rare cases low-grade MALT lymphoma are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal, disease.

Material and methods:
A total of 18 MALT lymphomas and 25 DLBCLs involving ocular adnexal sites were included in the study. All sections were analyzed immunohistochemically by two independent pathologists (ER, SH) using the following panel of antibodies: bcl-2, bcl-6, CD3, CD5, CD10, CD20, CD23, CD79α, cyclin D1, MUM-1 and Ki-67. Confirmed cases of DLBCL were categorized as either germinal centre B-cell-like (GCB) or non-GCB types according to the algorithm by Hans et al. Using LNA-based arrays from Exiqon, we performed global miRNA expression profiling of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The most differentially expressed microRNAs were confirmed by RT-qPCR analyses. Microarray processing was performed using the R environment.

Results:
Of the18 MALT patients (pts.) 15 pts. (83%) presented with Stage I lymphoma and 3 pts. (17%) had Stage IV. The 5-year overall survival for the entire population was 77%.
In the DLBCL group 13 pts. (52%) presented with Stage I lymphoma, 3 pts. (12%) Stage II lymphoma, 1 pt. (4%) Stage III and 8 pts. (32%) presented with Stage IV lymphoma. Nine of the DLBCLs were classified as GCB and 16 as non-GCB type. The 5-year overall survival for the entire group was only 13%.
Our miR arrays and confirmatory qPCR analysis revealed 43 miRNAs with significantly altered expression profiles (41 down- and 2 upregulated) in DLBCL compared to MALT lymphoma. Seven of the miRNAs down-regulated in DLBCL relative to MALT lymphoma showed enrichment for a direct transcriptional repression by the oncoprotein MYC. Supervised hieracical clustering analysis identified tree clusters: Cluster 1: MALT (high expression), cluster 2: DLBCL (intermediate expression), cluster 3 DLBCL (low expression). Thus, apparently the DLBCLs in cluster 2 seem to resemble MALT more than DLBCLs in cluster 3. We also report loss of miRNAs involved in the regulation of NFKB1 and DNA methyltransferases in DLBCL vs MALT.

Conclusion:
We conclude that fundamental differences in miRNA expression exist between ocular adnexal MALT lymphoma and DLBCL. Among the possible consequences of altered miRNA expression are increased NF-kB signaling and DNA hypermethylation. However, in line with the recent observations in gastric MALT/DLBCL transformation1, we suggest the differences may at least in part be caused by MYC transcriptional regulation of miRNAs in aggressive cases. The fraction of DLBCL that is reported to arise from MALT is exceedingly low. However, in the current study we find a group of DLBCLs (cluster 2), whose level of MYC regulated miRNA expression is intermediate between MALT (cluster 1) and DLBCL (cluster 3). Thus it could be speculated whether cluster 2 DLBCLs have developed secondary to a preceding MALT lesion, which, however was not detected by histology.

Disclosures:
No relevant conflicts of interest to declare.