The Effect Of HBV Infection On The Prognosis Of Diffuse Large B-cell Lymphoma And Its Role In Chemotherapy Resistance

Research BackgroundDiffuse large B cell lymphoma(DLBCL)is a highly heterogeneous group of aggressive tumors and the most common subtype of malignant lymphoma in adults.Hepatitis B virus(HBV)infection is a global health problem.Accumulating research evidence suggests that HBV infects B lymphocytes and is associated with a higher risk of developing DLBCL in countries where HBV is endemic.In recent years,there have been many clinical studies on the association between HBV infection and DLBCL,but the impact of HBV infection on the efficacy of treatment and prognosis of patients with DLBCL is still controversial.Compared with the chemotherapy-only(CHOP:cyclophosphamide,adriamycin,vincristine,prednisone)regimen,the immunochemotherapy regimen containing rituximab significantly improved the prognosis of patients with DLBCL,the prognostic impact of different HBV infection statuses on patients with DLBCL in different therapeutic contexts(chemotherapy alone,immunochemotherapy)has been less well-studied and needs to be further elucidated.The HBx antigen,encoded by X gene region,the smallest open reading frame in the HBV viral genome,may be a key molecule driving the increased malignant biological behaviors of DLBCL,but whether it plays a role in influencing the response to chemotherapy in DLBCL and the exact mechanism is unknown.Research Objectives1.To analyze and compare the differences in clinical characteristics between HBs Ag-positive and HBs Ag-negative DLBCL patients in our center.2.To investigate the effect of different HBV infection status on the prognosis of DLBCL patients in different therapeutic backgrounds(chemotherapy alone,immunochemotherapy).3.To investigate the role of HBx,a functional protein encoded by the X gene region in HBV,in DLBCL chemotherapy response and its mechanism.Research Methods1.Clinical data analysis: a total of 692 DLBCL patients diagnosed and treated in the First Hospital of Jilin University from July 2011 to July 2022 were included in this study,and all patients received ≥3 cycles of CHOP or R-CHOP-like regimens.The patients were grouped according to serum hepatitis B surface antigen(HBs Ag)status:the HBs Ag-positive group and the HBs Ag-negative group.We compared the baseline characteristics,pathological characteristics,and treatment characteristics by chi-square test,compared the differences in survival rates by the Kaplan-Meier method,analyzed the risk factors affecting the prognosis of patients with DLBCL by Cox regression model,and applied the NGS method to detect gene mutation profiles in the pathological tissues of DLBCL patients with different HBV infection status.2.Experimental methods: To investigate the role of HBV antigen HBx in DLBCL chemotherapy response and its mechanism,we constructed HBx lentiviral vectors to infect DLBCL cells,and applied flow cytometry and RT-q PCR to detect the transfection rate,and immunofluorescence to observe the localization of HBx protein in the cells.Cell viability was detected by CCK8,and apoptosis were detected by the Annexin V-APC /7-AAD.The corresponding genes were interfered or overexpressed.RT-q PCR and Western-blot methods were used to detect m RNA and protein expression levels in cell samples,and isolate cell nuclei and cytoplasmic proteins when necessary.Dual luciferase reporter gene assays were performed to clarify the regulatory relationship between transcription factors and the promoters of target genes.In vivo,transplanted tumor models were constructed by injecting tumor cells into the subaxillary of NOD-SCID mice and injecting chemotherapeutic drugs intraperitoneally.Evaluated the tumor inhibition rate by combining with the ultrasound performance and the change of the weight of the tumor.HE staining was used to clarify the morphology of the tumor tissue,immunohistochemistry staining was used to detect the expression and localization of proteins in tumor tissue samples,and Elisa method was used to detect the level of liver function in the blood serum.Research Results1.The serum HBs Ag positivity rate among DLBCL patients(n=692)in our center was 12.1%.HBs Ag-positive group had more frequent abnormal liver function(P =0.003),hypoalbuminemia(P < 0.001),incidence of >2 extranodal organs(P = 0.011),and spleen involvement(P <0.001)than the HBs Ag-negative group.When receiving the same kind of therapies,HBs Ag-positive patients had lower CR and ORR rates(P<0.05),in either the CHOP group or R-CHOP group.Among patients receiving RCHOP,the incidences of POD12 and POD24 were higher in the HBs Ag positive group than in the HBs Ag-negative group(P=0.018,P=0.029).2.Multifactorial Cox analysis showed that HBs Ag positivity was only associated with poorer OS(OS: HR [95% CI] = 2.511[1.214-5.192],P = 0.013)in the CHOP group.Whereas in the R-CHOP group,HBs Ag positivity was associated with both poorer OS and PFS(OS: HR [95% CI] = 1.672 [1.050-2.665],P = 0.030;PFS: HR [95%CI] =1.536 [1.013-2.331],P = 0.043).3.The NGS results of 180 patients with primary DLBCL showed HBs Ag-positive patients with DLBCL had a higher prevalence of mutations in MYC,ATM,PTPN6,and epigenetically regulated genes,which may affect several key pathways involved in lymphoma development,such as epigenetic regulation,DNA damage repair,BCR/NF-κB,and immune evasion.4.Successful construction of DLBCL cells stably expressing HBx(HBx-SUDHL-4,HBx-U2932)and control cells(GFP-SUDHL-4,GFP-U2932).HBx was expressed in the cytoplasm and nucleus of HBx-SUDHL-4,HBx-U2932 observed by immunofluorescence staining.CCK8 results showed that with the increased concentration of epirubicin and vincristine,the cell viability rate of both groups decreased.When in the same dosage group,the cell viability rate of the HBx-expressing group was higher than that of the control group.After applying the IC50 concentration of epirubicin and vincristine to treat for 48 hours respectively,the apoptosis rate of the HBx-expressing group was lower than that of the control group,and the expression levels of apoptotic proteins Cleaved Caspase 3 and Cleaved PARP-1 were lower.5.RT-q PCR and Western-blot results showed that XIAP m RNA and protein was highly expressed in HBx-SUDHL-4 and HBx-U2932 cells;transfection of XIAPspecific si RNA increased the sensitivity of HBx-SUDHL-4 and HBx-U2932 cells to epirubicin and vincristine.6.In vivo,in combination with the ultrasound performance and tumor weight changes of mice,HBx was able to attenuate the tumor inhibitory effects of epirubicin and vincristine.Immunohistochemistry staining results showed that tumor tissues in the HBx-expressing group had high expression of XIAP at baseline,and low expression of Cleaved Caspase 3 and Cleaved PARP-1 after chemotherapy.The ELISA and HE staining results of liver tissues indicated that liver function was in the normal range during chemotherapy in control and HBx-expressing group of homozygous mice.7.Compared with the control group,NF-κB p65 m RNA and protein levels were highly expressed in HBx-SUDHL-4 and HBx-U2932 cells.Combined with the results of NF-κB p65 phosphorylation level and NF-κB p65 distribution,all suggested that the NF-κB pathway was over-activated in the HBx-expressing group of DLBCL cells.Dual luciferase reporter assay indicated that overexpression of NF-κB p65 significantly enhanced XIAP promoter activity.After applying different concentrations of NF-κB entry inhibitor Bay11-7082 to treat HBx-SUDHL-4 and HBx-U2932 cells,RT-q PCR and Western-blot results showed that the XIAP expression level in HBx-expressing group was gradually decreased with the increased concentration of BAY 11-7082.Research Conclusions1.Patients with DLBCL with combined HBV infection have unique clinical features(more frequent extranodal involvement,higher incidence of abnormal liver function and hypoalbuminemia),prognostic features(reduced sensitivity to first-line therapy,higher rates of early progression,shorter OS and PFS),and genetic features(frequent mutations in MYC,ATM,PTPN6 and epigenetic regulatory genes)compared to the HBs Ag negative group.2.HBx,a functional protein encoded by the X gene region in HBV,upregulates XIAP expression and inhibits Caspase 3-mediated endogenous apoptosis by inducing overactivation of the NF-κB pathway,thereby promoting DLBCL resistance to epirubicin and vincristine,first-line chemotherapeutic agents.