The Effect Of Soluble PD-L1 On Growth Of Lewis Lung Carcinoma And The Clinical Implication Of SPD-L1 In Peripheral Blood Of Patients With Advanced EGFR Mutated Lung Adenocarcinoma

Lung cancer is one of the most common malignancies in the world,the incidence and mortality of lung cancer ranks first in malignant tumors.The immune escape mechanism of malignancies has become a hot topic in recent years.It has been found that costimulatory molecules play very important roles in immune escape of tumor cells.The programmed death molecule-1(programmed death 1,PD-1)is a member of the CD28/B7costimulatory superfamily.Programmed death ligand-1(PD-L1)is one of PD-1 liangds.PD-1/PD-L1 can mediate the negative stimulatory signal,inhibiting the function of T and B lymphocyte,which is of great significance in tumor immunity.Studies have shown that many costimulatory molecules not only exist in membrane-bound form,but also present widely in soluble form,such as sCD28,sOX40L,sTNFR and sCTLA-4 and so on.The biological significance of these soluble molecules and their relevance to clinical disease remains to be elucidated.A large number of clinical studies have found that the expression of some soluble costimulatory molecules may be useful in diagnosing malignancies or predicting prognosis of the disease,e.g.tumor tissue expression of PD-L1 can be used as a biomarker for the clinical efficacy of anti-PD-L1 antibody in certain tumors.Our previous clinical studies have found that elevated expression of serum level of soluble PD-L1(sPD-L1)in advanced lung cancer,which is closely correlated with stages of lung cancer and lymph node metastasis.However,there are few domestic and global studies regarding the use of animal models to study the influence of sPD-L1 on the growth of Lewis lung cancer and the clinical implication of the expression of PD-L1 in peripheral blood of patients with advanced epidermal growth factor receptor(EGFR)mutated lung adenocarcinoma.Therefore,in this project,we established Lewis lung carcinoma model by mice and injected sPD-L1Ig intraperitoneally into the mice to observe its effect on the growth of the tumor.In addition,we detected the expression of sPD-L1 in peripheral blood of patients with advanced EGFR mutated lung adenocarcinoma.The relationship of sPD-L1 with sex,age and TNM staging was observed.The value of s PD-L1 and cancer embryo antigen(CEA)in clinical evaluation of advanced EGFR mutated lung adenocarcinoma was evaluated by analyzing the receiver operating characteristic(ROC)curve.Part I The effect of soluble PD-L1 on Growth of Lewis lung carcinomaObjective To study the effect of different levels of soluble programmed death ligand-1(sPD-L1)on tumor formation and growth in lung cancer model of C57BL/6mice.Methods The expression of programmed death ligand 1(PD-L1)on the surface of Lewis lung cancer cell line and the expression of programmed death 1(PD-1)on the peripheral blood and spleen T lymphocyte membrane of C57BL/6 mice were determined by flow cytometry.We established Lewis lung cancer model by C57BL/6 mouse.sPD-L1Ig was administered intraperitoneally(sPD-L1Ig:2.5、5、10μg/mouse).sPD-L1control protein was injected intraperitoneally as control.The effect of different levels of soluble PD-L1 protein on growth of Lewis lung carcinoma was observed.The spleen T lymphocyte subsets were determined by flow cytometry on the 18th day after tumor cell inoculation.Results The expression of PD-L1 was observed on the surface of Lewis lung cancer cell line and the positive rate was 17.06%.The positive expression rate of PD-1 on spleen T lymphocytes membrane in C57BL/6 mice was 18.0%in contrast to that on T lymphocytes membrane from peripheral blood,which was 2.9%.Compared with the control group,the volume of Lewis lung carcinoma was larger in the C57BL/6 mice treated with 10μg/mouse(P<0.05),while the volume of Lewis lung carcinoma treated with 2.5μg/mouse and 5μg/mouse had no significant difference compared with control group(P>0.05).The CD4/CD8 ratios of spleen T lymphocytes in untreated mice and control mice were 1.92 and 1.87 respectively,while the CD4/CD8 ratio of spleen T lymphocytes in C57BL/6 mice treated with sPD-L1Ig 10μg/mouse was 1.57.Conclusions In vivo,a certain level of soluble PD-L1(10μg/mouse)could promote the growth of Lewis lung carcinoma in C57BL/6 mice.Part II The level of soluble PD-L1 and the clinical implication of sPD-L1 in peripheral blood of patients with advanced EGFR mutated lung adenocarcinomaObjective To detect the level of soluble programmed death ligand-1(s PD-L1)in peripheral blood of patients with advanced epidermal growth factor receptor(EGFR)mutated lung adenocarcinoma and to explore its clinical implication.Methods Seventy-two patients with EGFR mutated advanced lung adenocarcinoma(EGFR mutation group)treated with EGFR-tyrosine kinase inhibitor(TKI)from the Department of Respiratory Diseases in The Second Affiliated Hospital of Soochow University were enrolled in the study from May 2015 to July 2016.At the same time,thirty-one patients with advanced EGFR wild type lung adenocarcinoma treated with EGFR-TKI[diagnosed via mini specimens from bronchoscopy or transthoracic needle aspiration biopsy(TNAB),matching in sex,age and tumor stage with EGFR mutation group]were selected as controls(EGFR WT group).The levels of sPD-L1 in peripheral blood were measured by enzyme-linked immunosorbent assay(ELISA)kit.According to the clinical response of two-month’s EGFR-TKI treatment,all patients with advanced EGFR mutated lung adenocarcinoma was divided into two groups:the disease progression group(PD group,36 cases)and the disease control group(DC group,36 cases).The level of sPD-L1 in peripheral blood between the two groups was observed.The relationship between sPD-L1 level and sex,age and TNM staging in peripheral blood of patients with advanced EGFR mutated lung adenocarcinoma was analyzed.The value of serum sPD-L1and cancer embryo antigen(CEA)in clinical evaluation of patients with advanced EGFR mutated lung adenocarcinoma was studied by analyzing the receiver operating characteristic(ROC)curve.Results The levels of sPD-L1 in peripheral blood of EGFR mutation group and EGFR WT group were 0.75(0.15~2.78)ng/ml and 1.56(0.85~3.29)ng/ml respectively(P<0.001).In the EGFR mutation group,the levels of sPD-L1 in peripheral blood of PD group and DC group were 1.175(0.62~2.78)ng/ml and 0.625(0.15~2.27)ng/ml respectively(P<0.001).The expression of sPD-L1 in peripheral blood of patients with EGFR mutated advanced lung adenocarcinoma was correlated with lymph node metastasis and distant metastasis(χ~2=10.985,P<0.05;χ~2=4.662,P<0.05).The area under the ROC curve of peripheral blood sPD-L1 and CEA was 0.893(95%CI:0.830-0.956,P<0.001)and 0.745(95%CI:0.652-0.839,P<0.001)respectively.When the cutoff value of sPD-L1was set to 0.815ng/ml,the Youden index was the maximum.Using 0.815ng/ml as cutoff value,the sensitivity and specificity were 77.8%and 91.4%respectively.Conclusions The level of sPD-L1 in peripheral blood of patients with advanced EGFR mutated lung adenocarcinoma after EGFR-TKI treatment was lower than that of EGFR WT group,which suggests that sPD-L1 expression may depend on the regulation of EGFR signaling pathway.The level of sPD-L1 in patients with advanced EGFR mutated lung adenocarcinoma can reflect the clinical response to EGFR-TKI.In summary,the results of this study were as follows:(1)Soluble PD-L1 above certain threshold level could promote the growth of Lewis lung carcinoma of C57BL/6mice.(2)The level of sPD-L1 in patients with advanced EGFR mutated lung adenocarcinoma can reflect the clinical response to EGFR-TKI.