Gene Expression Profiling of Age-Related Epstein-Barr Virus (EBV)-Associated B-Cell Lymphoproliferative Disorder Uncovers Alterations in Immune and Inflammatory Genes: Possible Implications for Pathogenesis,

Age-related EBV-associated B-cell lymphoproliferative disorder (AR-EBLPD) is classified as a subtype of diffuse large cell lymphoma (DLBCL) according to the WHO classification. However, molecular genetic characterization of AR-EBLPD remains largely unknown. We studied expression profiles of 5 AR-EBLPD and 8 EB-negative DLBCL samples using the Agilent 44K human oligonucleotide microarray. Total RNA was extracted from fresh-frozen tumor samples. Each microarray slide was converted into datasets using the Agilent Micro Array Scanner and Feature extractions. Data was standardized with Z-scores. Differences in mRNA expression levels between two sample groups were calculated using a two-sided t-test. A total of 1973 probes showed a p-value less than 0.05 with less than a 25% false discovery rate (FDR). These probes included 1688 genes. The number of probes showing high expression in AR-EBLPD and EB-negative DLBCL was 804 (693 genes) and 1169 (995 genes), respectively. First, we selected the top 300 differentially expressed genes. Genes highly expressed in AR-EBLPD included IL6, TNFAIP3, HOPX, and SLAMF1. IL6 is known as a gene encoding a cytokine which functions in inflammation and the maturation of B lymphocytes, and TNFAIP3 is known as a negative regulatory gene of the NF-kB pathway. HOPX and SLAMF1 are reported as genes related to lymphocyte function or the immune system (Schwartzberg et al. Nature immunology 2009, Hawiger et al. Nature immunology 2011). For better characterization, we next performed Gene Ontology Analysis using the WEB-based GEne SeT AnaLysis Toolkit and found that categories of external stimulus and inflammatory responses were enriched in AR-EBLPD. The Kyoto Encyclopedia of Genes and Genomes (KEGG)-signaling analyses showed that pathways of the NOD-like receptor (p-value =1.30e-06), JAK-STAT (p-value =9.01e-06), and Toll-like receptor (p-value =0.0002) were characteristic of AR-EBLPD. These results implied that inflammation would be prominent in AR-EBLPD cases. For validation, we next performed Gene Set Enrichment Analysis (GSEA) using all the database of KEGG pathways (186 gene sets). Dominant gene sets in AR-EBLPD included the cytokine-cytokine receptor interaction [Normalized Enrichment Score (NES) =2.66, p-value<0.001], NOD-like receptor pathway (NES =2.26, p-value<0.001), TOLL-like receptor pathway (NES =2.14, p-value<0.001), and JAK-STAT pathway (NES =1.79, p-value<0.001). Since all the pathways were related to the NF-kB pathway, inflammatory responses were suggested to activate the NF-kB pathway or vice versa. For confirmation, we finally performed GSEA using gene sets of the NF-kB pathway, which were obtained from a gene set reported by an NIH group (Puente et al. Nature 2011) and 30 gene sets in the GSEA database, and found that the gene sets of the NF-kB pathway were enriched in AR-EBLPD (Figure 1). Our results suggested that the inflammatory and immune-related genes were enriched in AR-EBLPD and that activation of the genes may be associated with NF-kB activation. Aberrant immune and inflammatory responses could define the clinical presentations of AR-EBLPD cases.