PD-L1 Expression Of Pancreatic Cancer In Peripheral Blood And Tumor Tissue

【Background&Objectives】Background:Pancreatic cancer(PC)is a common malignant tumor,with lethal nature,early metastasis,and there is still a lack of effective treatments.Immunosuppression is one of the characteristics of Tumor micro-environment(TME),which is an important immunological cause for early metastasis and chemotherapy-insensitive/resistant of PC.Programmed death-ligand 1(PD-L1)/Programmed death-1(PD-1)signal pathway is a negative immune regulatory pathway for healthy population to avoid excessive autoimmune response.Many existing studies have found a significant increase of PD-L1/PD-1 expression in many types of malignant tumors.It is an important regulatory mechanism for tumor immune suppression.Membrane PD-L1(mPD-L1)and soluble PD-L1(sPD-L1)are the two forms of PD-L1.Recently PD-L1 monoclonal antibody anti-tumor drugs have just been approved by the US FDA for treatments of several cancers,and the results are satisfied.In PC,the existing literatures mainly focused on the high expression of PD-L1 in PC tissues and as a negative prognostic factor.sPD-L1 in peripheral blood and mPD-L1 expression on circulating tumor cells(CTC)of PC have no data.Objectives:The expression of sPD-L1 in peripheral blood of PC and its clinical significance;Summarized the distribution of PD-L1 and CD8+T cells in PC tissues,and find if there is the negative feedback between PD-L1 and CD8~+T cells;The relationship between sPD-L1 in peripheral blood and PD-L1,CD8~+T cells expression in PC tissues;Verified The expression of mPD-L1 on CTC surface of PC;The effect of surgical trauma on the early changes of sPD-L1.【Methods】1、sPD-L1:All the newly diagnosed pancreatic benign and malignant lesions,chronic pancreatitis,healthy control group of a total of 154 people,including 85 cases of PC,benign pancreas in 14 cases.Past history of no allergies,immune-related diseases,tumor history and acute disease infection is permitted.Peripheral serum was collected before operation,and the serum was collected in 24 hours after operation if thay have.The sPD-L1 was detected by double sandwich ELISA within 3 months after serum collection and IFN-γwas detected at the same time.Clinical data were collected for correlation analysis.Patient follow-up is currently underway.2、Immunohistochemistry(IHC)of PD-L1 in PC tissues:Select the surgical specimens sufficient,pathology type of pancreatic ductal adenocarcinoma specimens,parenchymal interface at the mesenchyme,continuous sections.The highest and lowest 14 pathological specimens in accordance with preoperative sPD-L1expression are selected.PD-L1 and CD8~+T cells were immunohistochemically stained.Currently supplementing the experiment is going on to expand the study sample.3、CTC&mPD-L1 on CTC:CTC enrichment:Each peripheral blood sample 5ml,EDTA anticoagulation,do not take the first tube blood after puncture.Within 6 hours the use of density gradient centrifugation is for peripheral blood mononuclear cells(PBMC)collecting.After the immunomagnetic beads negative filtrating of PBMC,screening CTC,and preparing for immunofluorescence(IF).IF:The cells nuclear were stained with DAPI and stained with CD45 monoclonal antibody to excluding PBMC.Pan-CK monoclonal antibody was used to screen tumor markers,and with PD-L1 monoclonal antibody there is four-color immunofluorescence.CTC:pan-CK(+),CD45(-),DAPI(+),with cells’nuclear is larger than PBMCs’.If PD-L1positive,stained(green)located in the cell membrane.4、statistics:All data would be analysis together if necessary.【Results】1、sPD-L1:sPD-L1 was significantly higher in PC serum than that in the healthy control group,but lower than that in the chronic pancreatitis group;sPD-L1 was significantly associated with tumor staging and distant metastasis;There were significant differences in the expression of sPD-L1 in pancreatic lesions of different pathological types;serum sPD-L1 and IFN-γlevels were consistent;sPD-L1 were significantly correlated with blood cells,pancreatic head tumors,jaundice,CA199 and so on;Regardless of benign and malignant lesions,sPD-L1 increased significantly within 24hours after surgical trauma.2、IHC:PD-L1 positive rate was 39.2%(11/28),CD8~+T cells 64.3%(18/28);There was no significant correlation between serum sPD-L1 and PD-L1 IHC positive rate in PC,which was consistent with the positive rate of CD8~+T cells;There was a negative feedback regulation of PD-L1 and CD8~+T cells in cancer tissues,and the spatial distribution according with the regulation;3、CTC&mPD-L1 on CTC:CTC was found only in patients with distant metastases of PC;Confirmed the presence of PD-L1 expression on CTC surface of PC.【Conclusions】sPD-L1 is highly expressed in patients with PC,especially with distant metastases,and there is PD-L1 expression on CTC surface of PC.PD-L1 abnormal expression in PC peripheral blood may be a promoting role for systemic immune suppression and CTC immune escape.The expression of PD-L1 in cancer tissue can promote the apoptosis of CD8~+T cells and the production of immunosuppressive environment in TME.The expression of PD-L1 was up-regulated within 24 hours after operation.The up-regulate expression of PD-L1 protect the body from excessive autoimmune response,but on the other side it is one of the possible risk factors for the malignant tumors,particularly those who can not be R0 resection of the surgical cases.The expression of PD-L1 and TILs in PC tissue,and sPD-L1,PD-L1 on CTC in the peripheral blood,were evaluated comprehensively,which could help to evaluate the immunosuppression status of PC patients.Precision Medicin for PC will benefit from this research.