Clinical Features Of Epstein–Barr Virus–Positive Diffuse Large B Cell Lymphoma And Roles Of Circular RNA In Regulating Cell Proliferation

Background:Epstein-Barr virus(EBV)infection occurs in 9%-15% of diffuse large B-cell lymphoma(DLBCL)and is associated with poor clinical outcome.Despite advances in clinical and molecular understanding of DLBCL,clinical and genomic features of EBV+DLBCL still remains unclear.MethodsTo illustrate clinical significances and molecular features of EBV+DLBCL,we assessed Epstein-Barr encodedRNA(EBER)by in-situ hybridization in tumor samples of 429 newly diagnosed DLBCL patients from a multi-center,phase 3,randomized,controlled trial(NCT01852435).Serum EBV DNA and serological tests were measured by quantitative PCR and enzyme-linked immunosorbent assay(ELISA).The efficacy of increased dose anthracycline was evaluated in DLBCL patients and in B-lymphoma cells.Genomic profiling was performed in 180 patients by whole exome/genome,targeted,orRNA-sequencing to identify the genomic features of EBV+DLBCL.Results46 of 429(10.7%)DLBCL patients were EBER positive and independently indicated inferior progression-free survival(PFS,hazard ratio: 2.58;95% confidence interval: 1.08-6.15;P =0.03)and overall survival(OS,hazard ratio: 3.71;95%confidence interval: 1.25-11.01;P =0.02).Serum EBV DNA showed correlation with tumor EBER and adverse clinical outcome.VCA IgA positivity was related to elevated LDH and intermediate-high/high-risk international prognostic index.Compared with EBER-negative patients,EBER-positive patients revealed a distinctive pattern of copy number alterations and highly prevalent chromosomal deletions involving MHC class?/?-mediated antigen presentation.Moreover,highly recurrent oncogenic mutations(TET2,DDX3 X,MYC,STAT3,TNFAIP3,TNFRSF14 and LYN),as well as dysregulation of mutations involving oncogenic pathways(Wnt,JAK-STAT,NF-?B and BCR signaling pathway)were also observed in EBER-positive patients.Increased dose of anthracycline may overcome adverse effect of EBV positivity on DLBCL patients and in vitro.ConclusionEBV contributed to tumor progression in DLBCL with distinct genomic and transcriptomic signatures and could be targeted by anthracycline dose intensification.BackgroundEpstein-Barr virus-positive diffuse large B-cell lymphoma(EBV+DLBCL)is characterized by poor prognosis and resistant to conventional chemotherapy.It is of great significance to explore the molecular mechanism of tumor progression and chemotherapy resistance in EBV+DLBCL for improving the therapeutic effect and overall survival of EBV+DLBCL.Increasing studies have showed that circRNAs play a vital role in regulating tumor growth,progression and drug resistance.However,the role of circRNAs and the underlying mechanisms in EBV+DLBCL remain largely unknown.Methods:Illumina Hi Seq sequencing was used to detect the sequence and expression of circRNA in tumor tissues from 8 cases of EBV-positive DLBCL and 5 cases of EBV-negative DLBCL.The expression of circRNA in tumor tissues of 100 patients with DLBCLs.By quantitative reverse transcription PCR in tumor,the relationship between the expression of circRNA and EBV infection,clinical characteristic and prognosis were further analyzed.The circular structure of circRNA was validated by Northern blotting,RNase R and actinomycin D treatment.UsingRNA pull down and luciferase assay,miRNA specifically binding to circRNA was identified.Gain-of-function experiments were conducted to investigate the biological functions of circRNA both in vitro and in vivo.The expression of important members of the signaling pathway was dected by Western blotting.Results:circEAF2 was downregulated in EBV+DLBCL tissues and cell lines,and its low expression was positively correlated to EBV+DLBCL progression and chemoresistance.Functionally,circEAF2 overexpression induced tumor cell apoptosis,inhibited cell proliferation and regulated epirubicin sensitivity both in vitro and in vivo.RNA pull down and luciferase assays revealed that EBV-encoded miR-BART19 was a direct target of circEAF2,which functioned as a sponge in regulating cell proliferation.Mechanistic analysis demonstrated that miR-BART19 specifically targeted the APC gene,and circEAF2/miR-BART19/APC suppressed the EBV+DLBCL proliferation and chemoresistance to epirubicin by inactivating the Wnt/?-catenin pathway.Conclusions:Our findings suggested that lower expression of circEAF2 was associated with aggressive tumor phenotypes and poor patient outcomes in EBV+DLBCL.circEAF2 inhibited EBV+DLBCL progression by acting as a sponge for miR-BART19 to regulate APC expression and the Wnt/?-catenin pathway,suggesting that circEAF2 might serve as a potential prognostic biomarker and therapeutic target for EBV+DLBCL.