Screening And Functional Studies On Genes Predicting The Efficacy Of Immunotherapy For Advanced Biliary Tract Cancer

Background and objectiveBiliary tract cancer(BTC)is a highly invasive malignant tumor.Due to lack of specific manifestations at the early stage and the propensity of invasion of adjacent organs,lymphatic and distant metastasis,most patients are often at the advanced stage before diagnosis and lose the chance of radical resection,resulting in a poor overall prognosis.Immune checkpoint inhibitors(ICIs),especially programmed cell death protein-1(PD-1)inhibitors,have been recognized as an effective option for a variety of tumors.However,the objective response rate of advanced BTC patients to ICIs treatment is unsatisfactory,and only a small number of patients get clinical benefits from ICIs treatment.Therefore,the discovery of biomarkers that can effectively predict the efficacy of immunotherapy and the accurate screening of the beneficiary population before treatment are the key issues to be addressed urgently in the development of BTC immunotherapy.This study aimed to explore the treatment effects and safety of PD-1 inhibitors in advanced BTC,to screen relevant genes that may predict the benefit of PD-1 inhibitor treatment in patients with advanced BTC,and to investigate the effects of key genes on PD-L1 expression and immune cells for elucidating their regulatory function.MethodsPart I:Efficacy and prognostic factors of PD-1 inhibitors in the treatment of advanced BTCThe clinical data of 92 patients with advanced BTC who received PD-1 inhibitor-based systemic therapy were retrospectively analyzed to explore the efficacy of different treatment options,possible prognostic risk factors and the response rate of different types of BTC to PD-1 inhibitors.Part II:Screening of key genes that may predict the efficacy of PD-1 inhibitors in advanced BTCThe samples of 92 patients with advanced BTC in the first part were screened through pathological evaluation and quality control of the sequencing platform,and 41 patients whose samples met the criteria were divided into durable clinical benefit group(DCB)and no clinical benefit group(NCB)according to the Response Evaluation Criteria in Solid Tumors(RECIST)Version 1.1.The expression of 289 immune-related genes in tumor tissues were detected through Nanostring sequencing platform and differentially-expressed genes between the DCB group and NCB group were screened out and further verified in tumor tissues using immunohistochemistry.Part III:Effects of high expression of S100A8 on PD-L1 expression and immune cell activation in BTCThe expression of S100A8 and PD-L1 in gallbladder cancer(GBC)cells and cholangiocarcinoma(CC)cells were analyzed by quantitative PCR(q PCR).The expression of S100A8 was overexpressed or knocked down by cell transfection in GBC cells,followed by investigation of the effects of S100A8 overexpression or knockdown on PD-L1 expression and activation of immune cells.LDH activity assay and IFN-γconcentration assay were carried out to investigate the killing effects of tumor cell-specific CD8~+cytotoxic T cells(CTL)on GBC cells with different S100A8 expression and the influence of PD-1 antibody on the killing effects.The killing effects of tumor cell-specific CD8~+CTL on GBC cells with different S100A8 expression were also tested in vivo.ResultsPart I:In this study group,intrahepatic cholangiocarcinoma(ICC),extrahepatic cholangiocarcinoma(ECC),and GBC were 34,27 and 31 cases respectively.After 6months of PD-1 inhibitor-based treatment,the ORR of GBC and ICC groups were 25.8%and 23.5%respectively,which were better than that in ECC group(0%),and the difference was statistically significant(P<0.05).The median progression-free survival(m PFS)and median overall survival(m OS)in the group of PD-1 inhibitor combined with targeted therapy were 5.0 months and 11.0 months respectively,which were greater than those in the group of PD-1 inhibitor monotherapy(2.5 months and 7.5 months),and the difference was statistically significant(P=0.007 and P=0.005);the m PFS and m OS in the group of PD-1 inhibitor combined with chemotherapy were not significantly different from those in the monotherapy group and combined targeted therapy group.Part II:Ten differentially-expressed genes were screened through Nanostring sequencing platform between the DCB group and NCB group.Nine of them(CTAG1B、CEACAM3、CXCL5、HLA-DQA2、CD19、S100A8、S100A9、G6PD、IL1β)were highly expressed and one(EGFR)was low expressed in the DCB group.Immunohistochemistry showed that the expression of S100A8 was significantly high in the DCB group(P<0.01)and was positively associated with the expression of PD-L1 in tumor samples(r=0.577,P<0.01).Part III:The expression of S100A8 and PD-L1 were significantly high in GBC and CC cells.In GBC cells,overexpression of S100A8 promoted the expression of PD-L1,while knockdown of S100A8 had the opposite effect.Knockdown of S100A8 significantly stimulated CD8~+T cell activation,while overexpression of S100A8 significantly inhibited CD8~+T cell activation.After adding PD-1 antibody,the killing effects of tumor cell-specific CD8~+CTL on GBC cells were enhanced significantly,especially on GBC cells with overexpression of S100A8.In vivo experiments,GBC cells with high expression of S100A8 can inhibit the killing effects of tumor cell-specific CD8~+CTL compared with GBC cells with low expression of S100A8.Conclusions1.For patients with advanced BTC,the efficacy of PD-1 inhibitor combined with targeted therapy was better than that of PD-1 inhibitor monotherapy.Compared with ECC patients,patients with GBC and ICC might obtain more benefits from PD-1 inhibitor-based systemic therapy.2.Compared with the NCB group,S100A8 was confirmed to be highly expressed in tumor tissues of the DCB group.And S100A8 was positively correlated with PD-L1 expression in the DCB group,suggesting that BTC patients with high S100A8 expression may benefit from PD-1 inhibitor therapy.3.S100A8 is significantly overexpressed in BTC cells,and overexpression of S100A8 ight promote BTC immune escape by promoting PD-L1 expression and inhibiting D8~+T cell activation.4.S100A8 may be a novel biomarker for predicting the efficacy of BTC immunotherapy r a target for tumor treatment.