Comprehensive Genomic Profiling of EBV-Positive Diffuse Large B-cell Lymphoma and the Expression and Clinicopathological Correlations of Some Related Genes

Abstract

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV+ DLBCL) is a rare type of lymphoma with a high incidence in elderly patients, poor drug response, and unfavorable prognosis. Despite advances in genomic profiling and precision medicine in DLBCL, EBV+ DLBCL remain poorly characterized and understood. We include 236 DLBCL patients for EBV-encoded mRNA (EBER) in situ hybridization detection and analyzed 9 EBV+ and 6 EBV negative cases by next-generation sequencing (NGS). We then performed fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to analyze chromosome rearrangements and gene expressions in 22 EBV+ and 30 EBV negative cases. The EBER results showed a 9.3% (22/236) positive rate. The NGS results revealed recurrent alterations in MYC and RHOA, components of apoptosis and NF-κB pathways. The most frequently mutated genes in EBV+ DLBCL were MYC (3/9; 33.3%), RHOA (3/9; 33.3%), PIM1 (2/9; 22.2%), MEF2B (2/9; 22.2%), MYD88 (2/9; 22.2%), and CD79B (2/9; 22.2%) compared with KMT2D (4/6; 66.7%), CREBBP (3/6; 50.0%), PIM1 (2/6; 33.3%), TNFAIP3 (2/6; 33.3%), and BCL2 (2/6; 33.3%) in EBV-negative DLBCL. MYC and KMT2D alterations stood out the most differently mutated genes between the two groups. FISH detection displayed a lower rearrangement rate in EBV+ cohort. Furthermore, KMT2D expression was highly expressed and associated with poor survival in both cohorts. MYC was only overexpressed and related to an inferior prognosis in the EBV+ DLBCL cohort. In summary, we depicted a distinct mutation profile for EBV+ and EBV-negative DLBCL and validated the differential expression of KMT2D and MYC with potential prognostic influence, thereby providing new perspectives into the pathogenesis and precision medicine of DLBCL.

Keywords: EBV-positive diffuse large B-cell lymphoma (EBV+ DLBCL); KMT2D; MYC; fluorescence in situ hybridizations; immunohistochemistry; next-generation sequencing.

Figure 1

Mutation profile in EBV+ and EBV- negative DLBCL groups. (A) The distribution and frequency of genetic alterations in nine EBV+ DLBCL and six EBV- negative DLBCL. The types of mutation and different clinical factors are labeled in different colors. (B) Gene mutation with potential targeted therapy. The frequent mutation genes in EBV+ and EBV- negative DLBCL cohorts and the potential targets highlighted by indicating the possible targeted therapy. (C) Pie chart showing the percentages of different types of somatic mutation in EBV+ DLBCL. (D) Mutation pathway distribution among EBV+ DLBCL specimens. Different color represents diverse mutation pathways and percentages.

Figure 2

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of EBV+ DLBCL. (A) Bioinformatic analysis for the top 20 GO enrichment in EBV+ DLBCL. (B) Bioinformatic analysis for the top 15 KEGG pathway enrichment in EBV+ DLBCL. (The P-value denoted the significance of GO terms enrichment in the genes and was adjusted by using Benjamini and Hochberg method to avoid false positives, and P < 0.05 was considered as significantly enriched).

Figure 3

FISH and IHC staining of EBV+ and EBV negative DLBCL specimens. Example of FISH performed on EBV+ and EBV negative DLBCL (A), FISH assay reveals rearrangement at the c-MYC (a), BCL2 (b) and BCL6 (c) loci using the dual-color break-apart rearrangement probes, break-apart signals in DLBCL nuclei are highlighted exemplarily by white arrows; increased copies of BCL6 by FISH (d). Immunohistochemistry staining for KMT2D and MYC in EBV+ and EBV negative DLBCL specimens (B), (a) Low expression of KMT2D (IHC, ×400); (b) High expression of KMT2D (IHC, ×400); (c) Low expression of MYC (IHC, ×400); (d) High expression of MYC (IHC, ×400). (C) Bar chart showing the percentages of KMT2D expression in EBV+ and EBV- negative DLBCL cohorts. (D) Bar chart showing the percentages of MYC expression in EBV+ and EBV negative DLBCL cohorts.