The Expression And Clinical Significance Of Soluble PD-L1 In Non-small Cell Lung Cancer Patients Receiving Radiotherapy

Objective: In recent years,PD-1/PD-L1 inhibitors,which activate immune system by inhibiting the PD-1/PD-L1 signaling pathway,have achieved good curative effect in the treatment of a variety of malignancies,and become the research hotspot.Detecting the expression level of PD-L1 in tumor cells has become an important method for predicting the treatment response of immune checkpoint blocking antibody.Combining PD-1/PD-L1 blockade with radiotherapy are becoming the latest treatment modality for non-small-cell lung cancer patients,but what is the proper timing and which subset of patients may benefit are still unclear.It is important to assess the expression of soluble PD-L1,which may be correlated with tumor tissue PD-L1 and patients' immunological status,in the serum of patients with lung cancer.The study intends to use ELISA method to detect the level of soluble PD-L1 in peripheral blood serum of patients with non-small cell lung cancer in different stages,and detect the expression level of sPD-L1 before,during and after radiotherapy dynamically.By analyzing its changing tendency and the association with patient's treatment responseand survival,we investigated the potential role of sPD-L1 expression level in the diagnosis,treatment responseprediction and prognosis of patients with NSCLC.To provide a reference for further basic research and clinical trials,we explored the timing of combination therapy of PD-1/PD-L1 inhibitors and radiotherapy for NSCLC patients.Method: We enrolled 136 NSCLC patients who had been diagnosed in histopathological biopsy with no EGFR gene-sensitive mutations,and randomly selected 61 healthy volunteers as subjects during the period between October 2013 and October 2016.According to tumor staging,NSCLC patients were divided into two groups: SBRT group and IMRT group.SBRT group patients(stage I-II)received cyber knife radiotherapy for local lesions,PTV prescription dose DT48-60 Gy,8-15Gy/f/d,1f/d,3-6f.The IMRT group patients(stage III)received intensity modulated radiotherapy,using the linear accelerator 6MV-X line,requiring 95% equal dose curves covering PTV,prescription dose DT60 Gy,2Gy/f/d,5f/w,and received synchronous chemotherapy and sequential chemotherapy,by docetaxel combined with cisplatin for adenocarcinoma,and by gemcitabine combined with cisplatin for squamous cell carcinoma.In the case of non-intervention,samples of serum,extracted from 5ml fasting blood in all subjects,were placed in-80? low temperature refrigerator.Subsequently,in the SBRT group,samples of serum were extracted in the same way on the 14 th day after the beginning of SBRT and the third month after the end of SBRT,respectively.In the IMRT group,samples of serum were extracted in the same way on the 14 th,28th and 42 nd days after the beginning of IMRT and the third month after the end of IMRT,respectively.The concentration of sPD-L1 in serum was detected by ELISA,as following: At first,each 0.1ml sample serum was placed inside the reaction hole,which had coated with PD-L1 antibody.After incubation and washing,the reaction was terminated when the enzymelabeled antibody and substrate liquid was added.The concentration of sPD-L1 was detected on a microplate reader,then recorded in computer.The follow-up included patients ' efficacy,disease-free time,total survival time and other informations.The objective response rate was evaluated by RECIST solid tumour curative effect criterion.The radiation toxicity was evaluated according to RTOG acute radiological injury grading standard and RTOG/EORTC advanced radiological injury grading standard.Statistics analysis: GraphPadPrism 6.0 software was used to draw a scatter plot and a trendline graph of sPD-L1 expression level.All the statistical data were analyzed by SPSS 22.0 software.Normal distribution data were described by ?±S,analysed by T test.Non-normal distribution data were described by M(Q1,Q3),analysed by nonparametric analysis.Measurement data were described by percentage,analysed by chi-squared test.Correlation analysis of Offset data were used rank-sum test.Analysis of survival factors wes used Kaplan–Meier or Cox Regression.A P-value of < 0.05 was considered be statistically significant.Results: The patients in healthy control group,whose average age was 63.7 years old(36-76 years old)and ratio of Male/Female was 2.1: 1,meeted the following requirements: no serious chronic diseases,no systemic immune disease,no infectious diseases,no trauma and acute infection within 3 months,no history of familial tumors.In NSCLC group,the patients' average age was 66.4 years old(36-85 years old),with the ratio of Male/Female 1.89: 1.The results of SBRT group: 29 patients with NSCLC were enrolled in SBRT group.The ratio of Male/Female was 2.63: 1.The average age was 72.5 years old.In all cases,5(17.2%)patients had complete remission,21(72.4%)patients had partial remission,2(6.9%)patients had stable disease,1(3.45%)patients had progressive disease.The rate of short-term efficacy(CR + PR)is 89.7%.The clinical benefit rate(CR + PR + SD)is 96.6%.The median overall survival(OS)is 21 months.The 3-year overall survival rate is 82.6%.The results of IMRT group: 107 patients with NSCLC were enrolled in the IMRT group.The ratio of Male/Female was 1.74: 1.The average age was 61.8 years old.According to the criterion of curative effect of solid tumor followup 0(0%)patients complete remission(CR),75(70.1%)patients partial remission(PR),32 patients(29.9%)stable disease(SD),2 patients(1.9%)progressive disease(PD).The rate of short-term efficacy(CR + PR)is 70.1%.The clinical benefit rate(CR + PR + SD)is 98.1%.The median overall survival(OS)is 17 months.The 3-year overall survival rate is 39.4%.The results of ELISA assay showed that the expression level of serum sPD-L1 in the initial state of health control group,SBRT Group and IMRT Group were 176.97 pg / ml(168.92,185.04),188.44 pg / ml(181.69,193.74)and 208.44pg/ml(196.95,223.45).The results of nonparametric analysis(Kruskal-Wallis H test and Mann-Whitney U test)showed that there was a statistically significant difference in the expression level of sPDL1 between the three groups and each two groups in initial state(P <0.01).All non-small cell lung cancer patients were divided into six subgroups according to the detailed tumor stage: ?A3,?B,?A,?A,?B and ?C.The expression levels of sPD-L1 in the subgroup were carried out non-parametric test,the results showed that there was gradually significant difference in each subgroup(P<0.01).It can be concluded that the later the stage of tumor,the higher the expression levels of sPD-L1.According to the expression levels of sPD-L1 in the initial state serum of all the patients,the ROC curve was drawn.Integral area of ROC was 0.870(P <0.01,95% CI 0.820-0.921).The value of Cutoff was 187.17pg/ml.The value of sensitivity was 0.787.The value of specificity was 0.779.The results suggest that detecting sPD-L1 expression has a referential significance for diagnosing NSCLC.According to SBRT and IMRT groups,the results of rank sum test of sPD-L1 expression data before and after treatment respectively showed that the expression level of sPD-L1 was decreased through radiotherapy.The change range of sPD-L1 expression was significantly different(P<0.01),and was related to the therapeutic effect.Results showed that the greater decrease of the expression level of sPD-L1,the efficacy is better.Some patients in the IMRT group had progression of disease after radiotherapy for 3 months,and their sPD-L1 expression level was also increased.Being the change of sPDL1 expression was significantly different(P<0.01),we concluded it was related to the prognosis for NSCLC.We calculated the change value of sPD-L1 expression level before and after radiotherapy in different groups,then arranged the rank and used the median as a cut-off point.The patients in each group are divided into the high change rate group and the low change rate group accordingly.,Kaplan-Meier method was used to analyze the survival rate between groups.The results showed that there was no significant difference in survival rate between SBRT subgroup(P = 0.926).There was significant difference in the survival rate between IMRT subgroups(P = 0.003),which indicated that high ?sPDL1 subgroup had the longer survival time.Multifactor survival analysis confirms that the ?sPD-L1 could serve as an independent prognostic factor(HR= 0.381,p = 0.033).Further analysis shows that the amplitude of reduction in sPD-L1 expression is increased in accordance with the irradiation dose,but the interferenceof time factor can't be ruled out.Conclusion: Through this retrospective study,we found that sPD-L1 is expressed in peripheral blood of both healthy person and non-small cell lung cancer patients.The expression level of sPD-L1 is higher in NSCLC patients than in healthy subjects,and the later tumor stage,the higher expression level.The expression level of sPD-L1 was decreased when patients were treated with radiotherapy(combined with or without chemotherapy).This trend was related to the treatment response and prognosis of patients with NSCLC,the greater the decline,the better response rate and survival.When the tumor progressed,the expression of sPD-L1 was increased,and this change was statistically significant.Therefore,sPD-L1 expression level in serum of NSCLC patients detected with Elisa method can provide some reference for the diagnosis of NSCLC.Monitoring the changes of SPD-L1 expression level dynamically can provide a tool for predicting treatment responseand prognosis in NSCLC.The value of its clinical significance needs to be verified by further research results.