Studies On The Clinical Significance Of BTK Expression And Synergistic Anti-tumor Effect Of BTK Inhibitor BGB-3111 Combinated With Bortezomib In Mantle Cell Lymphoma

Objective:In this study,we first detected the BTK expression in Mantle cell lymphoma(MCL)tissue and analyzed the correlation between BTK expression and clinic-pathological characteristics and prognosis of MCL patients.Then investigated the cytotoxicity of BGB-3111 in MCL cells,particularly combine with Bortexomib(BTZ),and explored its relative mechanisms.It is expected to provide a new idea of targeted drug combination therapy for improve MCL patient clinical outcome.Methods:1.The BTK expression in 32 cases of MCL was detected by immunohistochemical,the relationship between BTK and clinical features and prognosis of MCL patients were analyzed by SPSS 17.02.Three MCL cell lines were selected in cytology study.MTS and Compu Syn software were used to investigate the the cytotoxicity of BGB-3111 monotherapy and combined with bortezomib on the proliferation of MCL cells.3.PI staining and flow cytometry were used to detect the effect of BGB-3111 on the cell cycle of MCL cells.4.Annexin V / PI staining and flow cytometry were used to detect the effect of BGB-3111 and combined with bortezomib on apoptosis of MCL cells.5.The synergistic mechanism of BGB-3111 and bortezomib was evaluated by Western blotting.Results:1.Immunohistochemical staining showed that BTK protein was positive in MCL tissues and normal lymphoid tissues,but positive BTK was limit expressed in the mantle zone of all benign reactive lymphoid tissues and presented weak expression in germinal center cells.High strong positive expression level of BTK in MCL pathology could reach to 59.4%(19/32).2.Strong BTK expression had a statistically association with elevated Ki-67(χ2= 5.783,P = 0.019),and MIPI score(χ2 = 4.522,P = 0.038).no correlation with sex,age,B symptoms,bone marrow involvement,β2-microglobulin(β2-MG)and clinical stage(P﹥0.05).3.Kaplan-Meier prognosis analysis showed that strong positive BTK expression of patients had a poor progression-free survival(PFS)(P <0.05),but had no statistically significant difference between survival(OS)(P = 0.073).4.The results of univariate analysis showed that age ≧65 years(χ2= 11.348,P = 0.001),ECOG score ≧ 2 points(χ2 = 15.57,P <0.001),bone marrow involvement(χ2 = 4.138,P = 0.042),high positive BTK expression(χ2 = 4.69,P = 0.03),Ki-67 ≧30%(χ2 = 4.823,P = 0.028)and MIPI score ≧6(χ2 = 25.543,P<0.001)were all poor prognostic factors in MCL patients.However,multivariate Cox model analysis revealed that the strong positive expression of BTK can not be used as an independent prognostic factor for MCL patients due to the limitation of the sample size.5.BGB-3111 can inhibit Jeko-1,Rec-1,Z138 cells proliferation in time-and dose-dependent manner,and two drug combinations had significantly decreased cell viability in three MCL cell lines compared with single agent.6.PI staining and flow cytometry detection results showed that BGB-3111 treat Ments induced MCL cells arrest G0/G1 phase in dose-dependent manner.7.The results of apoptosis test showed that BGB-3111 could induce apoptosis in three MCL cells in a dose-dependent manner.The apoptosis rate was significantly increased after treatment with bortezomib,the difference was statistically significant(P < 0.01).8.Western blotting showed that BGB-3111 could reduce the phosphorylation of BTK protein and inhibit activity of NF-κB signaling pathway.Compared with BGB-3111 single drug,the BTZ significantly enhanced inhibited the phosphorylation activity of NF-κB pathway,down-regulated the expression of anti-apoptotic protein Bcl-2,and activated the cleavage of Caspase-9 and PARP.Conclusion:1.BTK protein was highly expressed in MCL tissues,and high expression of BTK was significantly associated with Ki-67 and MIPI scores.In addition,MCL patients with strongly BTK positive expression had a poor PFS,while those patients had no significant on OS compared with patients with BTK weakly expression.2.BGB-3111 inhibited the proliferation of MCL cells in time and dose-dependent manner,and induced MCL cell arrest in G0 / G1 phase.3.BGB-3111 combined with BTZ could synergistically inhibition of MCL proliferation and induction of apoptosis on MCL cells.4.BGB-3111 combined with BTZ synergistic mechanism may be related to the activation of NF-κB signaling pathway and the activation of apoptotic signaling pathway.