Analysis Of Clinical Features And Next Generation Sequencing Of Epstein–Barr Virus–positive Diffuse Large B- Cell Lymphoma

ObjectiveDiffuse large B-cell lymphoma(DLBCL)is the most common subtype of non-Hodgkin's lymphoma,and Epstein-Barr virus-positive DLBCL(EBV~+DLBCL)is a new subtype of DLBCL,with obvious clinical characteristics and poor outcome.This study was focused on the clinical characteristics,outcomes and molecular changes in EBV~+DLBCL.MethodsAll 600 cases enrolled in this study were from 16 centers in China between March2013 and June 2017.Inclusion criteria:newly diagnosed DLBCL;older than 16 years old;and with complete medical history information.Young patients(ages?60y)were randomized divided to three treatment groups(R-CHOP50,R-CEOP70,R-CEOP90)according to the dose of doxorubicin,while dividing the old patients into two groups(R-CHOP50,R-CEOP70).Epstein-Barr encoded RNAs-1(EBER)in situ hybridization was performed on 262 cases to detect EBV infection;quantitative PCR(qPCR)were performed on 192 cases in pretreated whole blood and serum for EBV DNA copies,and enzyme-linked immunosorbent assay(ELISA)was performed on 256 cases to test the specific antibodies of EBV with IgM,IgA and IgG classes.Clinical feature,overall survival(OS)and progress free survival(PFS)analyses were performed using SPSS software,version 22.0.Whole-genome/exome sequencing(WGS/WES)was performed on 122 patients,and targeted sequencing was performed on 75 patients.Gene mutation frequency and KEGG pathway analysis were performed according to EBV infected status.Results1.A total of 25 cases of EBER positivity were found in 262 cases(25/262,9.54%).EBV DNA was tested in pretreatment serum and whole blood(WB)from 192 patients,and 40 cases were identified as positive in whole blood(40/192,20.83%),and 15 cases were identified as positive in serum(15/192,7.81%).2.No significant differences were found in the clinical features between patients patients with EBV DNA positive and EBV DNA negative,as well as patients with EBER positive and EBER negative.EA IgG~+DLBCL more likely occurred in young women(p=0.0406,p=0.0406),EBNA IgG~+was predominantly observed in non-GCB phenotypic DLBCL patients(p=0.0323).VCA IgA~+DLBCL patients tend to accompany with advanced AA stage,higher IPI scores and elevate LDH level(p=0.0204,p=0.0156,p=0.0027).3.DLBCL patients with EBER~+had a worse prognosis than EBER~-(average survival:33.645 months vs.47.52 months,PFS:p=0.0020,OS:p<0.0001);Compared to DLBCL patients with EBV DNA negative(WB),the positive patients showed no significant different prognosis among all patients,but inferior survival in old(PFS,p=0.0274,OS:p=0.0259).Significantly decreased PFS was only found in young group with EBV DNA positive DLBCL(serum),VCA IgA~+DLBCL(p=0.004,p=0.042).What's more young group with EBNA IgG~+DLBCL showed an increased tendency in PFS,compared to the negative.In total,DLBCL patients with EBER~+,EBNA IgG~+or VCA IgA~+all had a better prognosis in the R-CEOP-90 treatment group than in the R-CHOP-50/R-CEOP-70 group.4.Different gene mutations analysis between EBV~+DLBCL and EBV~-DLBCL was performed in genes which mutation frequency was more than 5%by next generation sequencing and accepted as associated with DLBCL,significant results were as followed:PIK3CA:27.3%VS 4.6%,p=0.0016;TNFAIP3:18.2%VS 5.1%,P=0.0427;PIK3CD:18.2%VS 5.1%,p=0.0319.Some significant different pathways(NOTCH:36.36%VS 12.57%,p=0.0081;JAK-STAT:45.45%VS 18.29%,p=0.0146;BCR:22.73%VS 6.29%,p=0.0209;NF-?b:36.36%VS 10.29%,p=0.0029)were noted by KEGG analysis.ConclusionEBV~+DLBCL and EBV~-DLBCL were significantly different not only in clinical characteristics and survival,but also in some molecular pathways(NOTCH,JAK-STAT,NF-?b,BCR).Increasing dose of doxorubicin may overcome the poor outcomes in the EBV~+DLBCL.