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.