| Objective To evaluate the the expression levels of negative co-stimulatory molecules soluble programmed death-1(sPD-1)and soluble programmed death-ligand 1(sPD-L1)in ascites of patients with hepatocellular carcinoma(HCC)and to explore its clinical significance.To evaluate the expression of sPD-1 and sPD-L1 in serum of HCC patients and then explore its correlation with the expression level in ascites.The expression levels of sPD-L1 in the culture medium of human hepatocellular carcinoma cells was analyzed to analyze the possible sources of sPD-L1.Thus to provide experimental basis and theoretical basis for the further study of anti-PD-L1 treatment of HCC by interventional route.Materials and methods 1.The source of specimen.Forty cases of HCC patients with ascites were selected as the experimental group.All patients were pathologically or clinically diagnosed as HCC.Refering to “Specifications for diagnosis and treatment of primary liver cancer(2017 edition)”,the experimental group were divided into two subgroups:fifteen in the early HCC group(clinical stage I and II)and twenty five in the advanced HCC group(clinical stage III and IV).Twenty patients with chronic hepatitis B cirrhosis complicated with ascites were selected as control group.Three types of human hepatocellular carcinoma cell lines were cultured in vitro,including SMMC7721,HepG2 and Huh-7.The culture medium supernatant was analyzed as hepatocellular carcinoma cell lines group.And the blank culture medium which did not have any kinds of human hepatocellular carcinoma cell lines was arranged into blank control group.2.Specimen collecting.Ascites and peripheral blood serums were collected from the experimental group and the control group respectively.Among them,there were forty samples of ascites and forty samples of peripheral blood serum in the experimental group.In the control group,there were twenty samples of ascites and twenty samples of peripheral blood serum.The supernatants of the above three types of human hepatoma cell strain culture fluids were collected.3.Specimen testing.Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentrations of sPD-1 and sPD-L1 in the clinical specimens and the concentration of sPD-L1 in the culture fluid.4.Data collecting.Patient’s age,gender and history of liver surgery were observed and recored.The data of blood routine index,liver function index,tumor index,hemagglutination index were collected on the same day or within three days before and after the specimen was taken.The data of Child-Pugh score,clinical stage,BCLC stage and ECOG score were also collected.Enhanced CT or MRI scan were performed on the experimental group patients within one month before and after the sample collecting.According to the enhanced CT or MRI,the size of the tumor,the extent of the tumor,the number of tumors,portal vein invasion and distant metastasis were also assessed.5.Statistical Analysis.SPSS 24.0 statistical software was used for data analysis.Measured data were expressed as mean±standard((x|-)±s)deviation and compared with t-test or Mann-Whitney U test.Count data adoption rate(%)was expressed and compared with chi-square test.Correlations between variables were analyzed using Pearson correlation analysis or Spearman correlation analysis.Receiver operator characteristic curve(ROC curve)was used to evaluate the sensitivity of sPD-1 and sPD-L1 expression levels in ascites in the diagnosis of HCC.And P<0.05 was considered statistically significant.Results 1.Clinical results.In the experimental group,there were 31 males and 9 females,aged 39-74(60.03±10.64)years.The BCLC stage consisted of 6 patients in stage A,12 patients in stage B,and 22 patients in stage C.The Child-Pugh classification was grade A in 17 patients,grade B in 17 patients,and grade C in 6 patients.The average lesion diameter was(5.57±3.08)cm.The control group including 12 males and 8 females,aged from 42-82 years(61.75±12.20 years).For the Child-Pugh classification,there was 11 patients in grade B,and 9 patients in grade C.2.Specimen testing results.2.1 Expression levels of sPD-1 and sPD-L1 in ascites.The concentrations of sPD-1 and sPD-L1 in the ascites were 0.3895±0.1103(ng/ml)and 0.7599±0.3538(ng/ml)respectively in the experimental group,while 0.3491±0.0786(ng/ml)and 0.3521±0.2008(ng/ml)respectively in the control group.The sPD-1 and sPD-L1 concentrations in the experimental group were significantly higher than those in the control group,the difference was statistically significant(P<0.05).2.2 Expression levels of sPD-1 and sPD-L1 in serum.The serum concentrations of sPD-1 and sPD-L1 in the experimental group were 0.4408±0.1158(ng/ml)and 0.9067±0.23206(ng/ml)respectively;in the control group they were 0.3688±0.0480(ng/ml)and 0.7525±0.0888(ng/ml)respectively.The concentrations of sPD-1 and sPD-L1 in the experimental group were significantly higher than those in the control group,and the difference was statistically significant(P<0.05).2.3 Expression level of sPD-L1 in the culture medium supernatant.The concentrations of s PD-L1 in SMMC7721,HepG2 and Huh-7 culture medium were 0.2890(ng/ml),0.3753(ng/ml)and 0.4257(ng/ml)respectively.The concentration of sPD-L1 in the blank control sample was 0.1563(ng/ml).The expression level of sPD-L1 in human hepatocellular carcinoma cell lines culture medium in vitro was significantly higher than that of blank control sample,and the difference was statistically significant(P<0.05).3.Subgroup analysis of the experimental group.The concentrations of sPD-1 and sPD-L1 in patients of early HCC group was 0.3367±0.0221(ng/ml)and 0.5902±0.1152(ng/ml)in ascites,and 0.3768±0.0549(ng/ml)and 0.8023±0.2037(ng/ml)in serum respectively.The concentrations of sPD-1 and sPD-L1 in advanced HCC group was 0.4153±0.0884(ng/ml)and 0.8362±0.4149(ng/ml)in ascites and 0.4780±0.1301(ng/ml)and 0.9596±0.2261(ng/ml)in serum respectively.The levels of sPD-1 and sPD-L1 expression in ascites and serum of patients with advanced HCC were significantly higher than those of early HCC,and the difference was statistically significant(P<0.05).4.Correlation between s PD-1 and sPD-L1 expression levels in ascites and the clinical data of patients in the experimental group.The expression of sPD-1 and sPD-L1 in ascites of patients with HCC was positively correlated with BCLC stage,Child-Pugh grade,portal vein invasion,distant metastasis and clinical stage(P<0.05).There was no significant correlation with age,gender,surgical history,ECOG score,baseline lesion diameter,tumor size or tumor diameter(P>0.05).5.Correlation between s PD-1 and sPD-L1 expression levels in ascites and laboratory tests in the experimental group.The expression levels of sPD-1 in ascites of HCC patients was positively correlated with the levels of PLR,WBC,APTT,AST,ALT,AFP,PIVKA-II,PT,and PT-INR(P<0.05),and was negatively correlated with the levels of ALB and PAB(P<0.05).The expression level of sPD-L1 was positively correlated with the levels of NLR,PT-INR,PT,APTT,PIVKA-II,AST,ALT and AFP(P<0.05).The expression levels of sPD-1 and sPD-L1 were not significantly correlated with the expression levels of LY,NE,RBC,PLT,T-BIL,AST/ALT,γ-GT,ALP,GLB,ALB/GLB,CRP and TT(P>0.05).6.Correlation between sPD-1/sPD-L1 expression levels in ascites and serum of HCC patients.The expression level of sPD-1/sPD-L1 in ascites was positively correlated with the expression level of sPD-1/sPD-L1 in serum(P<0.05).The expression level of sPD-L1 in ascites/serum was positively correlated with the sPD-1 expression levels(P<0.05).7.Receiver operator characteristic curve.The area under the curve corresponding to sPD-1 in ascites was 0.694(P=0.015),the 95% confidence interval(95% CI)was(0.539-0.848),and the optimal cut-off value for diagnosis of HCC was 0.3140(ng/ml),the sensitivity was 80.0% and the specificity was 55.0%.The area under the curve corresponding to sPD-L1 in ascites was 0.841(P<0.001),the 95% CI was(0.740-0.943),the optimal cut-off value for diagnosis of HCC was 0.5538(ng/ml),the sensitivity was 77.5% and the specificity was 85.0%.Conclusions This initial study revealed: 1.sPD-1 and sPD-L1 were highly expressed in ascites and peripheral blood of HCC patients.2.The expression of sPD-L1 in culture medium of human hepatocellular carcinoma cell lines was positive,and this suggested that hepatocellular carcinoma cells may be an important source of sPD-L1.3.The expression of sPD-1 and sPD-L1 in ascites and serum of patients with advanced HCC were significantly higher than those of patients with early stage HCC.4.The expression of sPD-1 and sPD-L1 in ascites of HCC patients was correlated with clinical indicators such as BCLC stage,Child-Pugh classification and portal vein invasion,as well as some blood inflammation indexes,liver function indexes,hemagglutination indexes and tumor indexes,indicating that it had certain clinical value in assessing tumor invasiveness,impaired liver function,and inflammatory state of the body.5.For HCC patients,the expression of sPD-1/sPD-L1 in ascites was positively correlated with the expression level of sPD-1/sPD-L1 in serum,and the expression of sPD-L1 in ascites and serum was positively correlated with the expression of sPD-1.6.The detection of sPD-1 and sPD-L1 in ascites had potential clinical value in the screening and diagnosis of HCC. |
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