The Expression And Functional Mechanisms Of PD-L1 In Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is a malignancy tumor.Patients with HCC had poor prognoses.China is a high incidence area.The number of patients with HCC in China is more than 55%in the world.Moreover,HCC has diversified pathogenic factors.Tumor recurrence is the primary cause of death.Therefore,seeking risk factors of HCC recurrence has important clinical significance.Intervening and treatment these risk factors is an important way to prevent HCC recurrence and improve overall survival.Programmed cell death 1(PD1)is the inhibitory receptor of T cells and activated B cells.As a family member of CD28,PD1 can be interacted with B7family ligands(PD-L1 and PD-L2).These ligands are pivotal attacked molecular target of T cells.Nowadays,some studies reported that the expression of PD-L1 or PD-L2 is significantly associated with cancer patients'survival.And some studies also reported that anti-PD1 inhibitor is effective for some patients.However,only a few studies have been reported about association between the expression of PD-L1/L2 and HCC patients'prognoses.In addition,adaptive immunity cells in carcinoma foci and paratumor take part in tumor immune microenviroment.Study revealed that the expression of side population cells,such as CD4~+T lymphocyte and CD8~+cytotoxic T lymphocyte,is associated with colorectal cancer patients'recurrence rate and overall survival.Among research field of HCC,no study is reported about the clinical application value of immunoreactive score.Therefore,it is important to analyze the role of immunoreactive score in assessing patients'disease-free survival and overall survival.And third,very few studies discussed association between the expression of PD-L1 and tumorigenicity.Based on these research backgrounds,this study aims to collect HCC patients'clinical pathology and prognostic analysis data,analyze the expression of PD-L1/L2,CD4~+T lymphocyte and CD8~+cytotoxic T lymphocyte in carcinoma foci and paratumor by immunohistochemical analysis,and then investigate the associations among these parameters.These works hopes to explore the possibility of PD-L1/L2,CD4~+T lymphocyte or CD8~+cytotoxic T lymphocyte as a prognostic marker for patients with HCC after curative resection.At the same time,we perform basic experiment to investigate the role of PD-L1overexpression or knockdown in HCC cell biological behavior.These results will provide some theoretical foundation for further experimental research in vivo and clinical application.Part I Prognostic value of programmed death-ligand 1 expression inpatients with solid tumors:meta-analysisBackground:Programmed death-ligand 1(PD-L1)is thought to play a critical role in immune escape by cancer,but whether PD-L1 expression can influence prognosis of patients with solid tumors is controversial.Therefore,we meta-analyzed available data on whether PD-L1 expression correlates with overall survival(OS)in such patients.Methods:PubMed,EMBASE and other databases were systematically searched for cohort or case-control studies examining the possible correlation between PD-L1 expression and OS of patients with solid tumors.OS was compared between patients positive or negative for PD-L1 expression using scatter plots,and subgroup analyses were performed based on tumor type and patient characteristics.Results:Data from 59 studies involving 20,004 patients with solid tumors were meta-analyzed.The median percentage of tumors positive for PD-L1 was30.1%.OS was significantly lower in PD-L1-positive patients than in PD-L1-negative patients at 1 year(P=0.039),3 years(P<0.001)and 5 years(P<0.001).The risk ratios of OS(and associated 95%confidence intervals)were 2.02(1.56-2.60)at 1 year,1.57(1.34-1.83)at 3 years and 1.43(1.24-1.64)at 5 years.Similar results were obtained in subgroup analyses based on patient ethnicity or tumor type.Among the seven included studies for HCC,the risk ratios of OS(and associated 95%confidence intervals)were 1.87(1.01-3.46)at1 year,1.40(0.92-2.15)at 3 years and 1.58(1.11-2.25)at 5 years.Conclusion:The available evidence suggests that PD-L1 expression negatively affects the prognosis of patients with solid tumors.However,larger prospective studies are needed to verify this,especially for different tumor types.Testing for PD-L1 expression may aid in treating solid tumors and predicting prognosis for affected patients.Part II Association between the expression of PD-L1/L2,infiltration ofCD4~+/CD8~+T lymphocyte and HCC patients'prognosesAims:To investigate the association between the expression of PD-L1/L2,infiltration of CD4~+/CD8~+T lymphocyte and HCC patients'prognoses.Methods:qRT-PCR and Western blot was used to detect the expression of PD-L1/L2 in carcinoma foci and paratumor.The expression of PD-L1/L2 and infiltration of CD4~+/CD8~+T lymphocyte was detected by immunohistochemical analyses of HCC tissues.Results:The mRNA and protein expression of PD-L1 and PD-L2 in HCC tissue was significantly higher than those in paratumor.High expression of PD-L1 or PD-L2 was associated with several poor clinicopathologic factors,such as multiple nodules,Edmondson-Steiner stage III/IV,and BCLC stage C.Patients with high expression of PD-L1 had significantly poor overall survival than those with low expression(P=0.036).However,patients with high or low expression of PD-L1 had similar disease-free survival(P=0.095).Moreover,patients with high or low expression of PD-L2 had similar overall survival or disease-free survival(all P>0.05).Patients with high infiltration of CD4~+T lymphocyte had significantly higher overall survival and disease-free survival than those with low infiltration(P=0.015,P=0.038).Also,patients with high infiltration of CD8~+T lymphocyte had significantly higher overall survival and disease-free survival than those with low infiltration(P=0.033,P=0.047).Multivariate analysis found tumor diameter equal or more than 5 cm,multiple nodules,microvascular invasion,incomplete/absent tumor capsule,preoperative or postoperative alpha fetoprotein equal or more than 400 ng/ml,Edmondson-Steiner stage III/IV,BCLC stage C,tumor recurrence,and higher expression of PD-L1 were risk factors of deaths,while high infiltration of CD4~+T lymphocyte,PD-L1 and PD-L2 simultaneously low expression were protective factors of deaths.In addition,tumor diameter equal or more than 5 cm,multiple nodules,microvascular invasion,incomplete/absent tumor capsule,preoperative or postoperative alpha fetoprotein equal or more than 400 ng/ml,and BCLC stage C were risk factors of tumor recurrence,while PD-L1 and PD-L2 simultaneously low expression was protective factors of tumor recurrence.In addition,chi square test found low expression of PD-L1 and high infiltration of CD4~+T lymphocyte had significantly correlation(P=0.010).Conclusion:High expression of PD-L1 and PD-L2 are associated with several clinicopathologic characteristics.High expression of PD-L1 and PD-L2are risk factors of tumor recurrence and death among patients with HCC after resection.Moreover,low expression of PD-L1 and high infiltration of CD4~+T lymphocyte had significantly correlation.Part III Research about the role of PD-L1 in HCC malignant biologicalbehaviorAims:To investigate the function of PD-L1 in HCC malignant biological behavior in vitro and in vivo.Methods:The expression of PD-L1 in HCCLM3,MHCC97-H,HepG2,and Huh7 cell lines was detected by PCR and Western blot.And then we use the cell line which has the highest expression of PD-L1 in the following experiment.The expression of PD-L1 in Huh7 cell line was knockdown or overexpression.In order to observe the influence of the expression of PD-L1 and anti-PD-L1treatment to tumorigenicity,tumor formation in nude mice was performed.Results:The expression of PD-L1 can be detected in HCCLM3,MHCC97-H,Hep G2,or Huh7 cell lines.And HepG2 cell line has the highest expression of PD-L1.PD-L1 overexpression can enhance tumor cell proliferation in vitro and tumorigenicity in vivo.And anti-PD-L1 treatment can reduce the rate of tumorigenicity in vivo.However,among those mouses injection with PD-L1knockdown cells,such results were not observed.Conclusion:All four HCC cell lines have PD-L1 expression.The expression of PD-L1 was associated with the ability of tumor cell proliferation in vitro and tumorigenicity in vivo.