Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

Abstract

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

Fig 1. (A) RNA-Seq technology reveals the presence of EBV and of other viruses. In particular, 5/20 cases contain human herpesvirus 5 (CMV), 4/20 human herpesvirus 8 (KSHV), and 1/20 human T-lymphotropic virus 1 (HTLV-1). (B) Immunohistochemical evaluation demonstrates the presence of CMV in the stromal cells in the adjacent reactive lymphoid tissue. CMV stain, Original Magnification (O.M.): 40x. (C) KSHV positivity is shown, respectively in few neoplastic cells and in the endothelial cells within the neoplastic proliferation. LANA-1 (LN53 antibody), O.M.: 40x; (D) LANA-1 (AT4C11 antibody) O.M.: 40x.

Fig 2. (A) Unsupervised hierarchical clustering of expressed EBV genes demonstrates a diversity of non-canonical latency-associated gene expression programs with a subset of viral episome initiating lytic reactivation as indicated by expression of genes corresponding to the lytic program. (B) LMP-2A is expressed by 40 to 50% of neoplastic cells. LMP-2A stain, O.M.: 40x; (C) LMP-2A expression is identified in a proportion of neoplastic cells ranging from 20 to 30%. LMP-2A stain, O.M.: 40x; (D) BZLF1/ZEBRA positivity is expressed by 5 to 10% of neoplastic cells. BZLF1/ZEBRA stain, O.M.: 40x; (E) BZLF1/ZEBRA expression is detected in few neoplastic cells. BZLF1/ZEBRA stain, O.M.: 40x; (F) BMRF-1/Ea-D expression is observed in 50% of neoplastic cells. BMRF-1/Ea-D stain, O.M.: 40x; (G) BMRF-1/Ea-D protein expression in 5% to 10% of neoplastic cells is shown. BMRF-1/Ea-D stain, O.M.: 40x; (H) BHRF-1/Ea-R staining is found in 60% of neoplastic cells. BHRF-1/Ea-R stain, O.M.: 40x; (I) BHRF-1/Ea-R is expressed in 10% of neoplastic cells. BHRF-1/Ea-R stain, O.M.: 40x.

Fig 3. (A) The presence of mutations in genes previously described in BL is reported, including MYC (50%), DDX3X (35%), ID3 (30%), ARID1A (25%), RHOA (20%), TCF3 and TP53 (15%), and CCND3 1/20 (5%). In addition, a new mutation is shown, involving CCNF and detected in 20% of the cases. (B) Bar plot showing the frequency comparison of virus presence and driver mutations between endemic and sporadic BL. For each comparison we report the p-value associated with rejecting the null hypothesis of equal eBL and sBL prevalences. (C) Distribution of mutations in 5 driver genes. Red points indicate endemic BL, while blue points the sporadic ones.

Fig 4. (A) the dendrogram classifies the samples into EBV-positive and EBV-negative BL independently on the specific subtype with an accuracy of 96% (45/47). (B) GSEA C2 analysis on genes differentially expressed between EBV-positive and EBV-negative cases detects a significant enrichment for the LMP-1 gene set signature. GSEA: gene set enrichment analysis.