Association Study Of Gene Polymorphisms In CD206/CD68 And Their Expression On Macrophages With Hepatocellular Carcinoma Pathogenesis And Prognosis

Background: Hepatocellular carcinoma(HCC)was one of the most lethal malignancy ranking as the fifth most common cancer with serious social burdens worldwide.There are numerous HCC patients in Asia.Currently,the morbidity and mortality of HCC are increasing in China.Although the basic and clinical research on HCC have made a lot of exciting results,the incidence of advanced HCC was increasing with dismal prognosis as these cases were associated with multiple metastasis.Recurrence after surgery also challenges us.Therefore,looking for more potential biomarkers and therapeutic targets will be helpful to early diagnosis and treatment for HCC patients.As a usual virus-associated cancer,HCC is considered to be closely linked to chronic infection,especially chronic inflammation with Hepatitis virus B which plays a critical role in the occurrence and development of tumor through continuous expression of cytokines and recruitment of immune cells to the liver.It is well known that genetic factors play an important role in the occurrence and development of HCC,of which single nucleotide polymorphism is one of the main form of gene polymorphism.Its frequency is more than 1% in the population.SNP can reflect the genetic differences among individuals and regulate gene expression,resulting in influencing the individual susceptibility to certain diseases.Currently,SNP has been widely applied to in the study of human diseases and tumor-related SNP is a typical example for the individual susceptibility to cancer.Tumor-associated macrophages(TAMs)are widely documented as a fundamental cellular component within the tumor immune microenvironment.There is extensive research on TAMs in the field of tumor.As pivotal mediators in the inflammatory response,macrophagescould generally be polarized into two principal subtypes: classically activated macrophages(M1)and alternatively activated macrophages(M2).Generally,TAMs are considered as M2-like macrophages,as a result,they pose biomarkers and pro-tumor characteristics similar to M2-like macrophages.However,M1 and M2 macrophages merely represent the two extremes of a continuum of functional status.Not surprisingly,TAMs are capable of reversibly transforming between M1 and M2 phenotypes,displaying functional plasticity.The complicated biological features of TAMsbring huge challenges in clinical research and practice.Nowadays,the roles of TAMs in the occurrence and development of primary HCC still remain incompletelyelucidated.CD68 iscommonly considered a pan-macrophage marker and a kind ofmacrophage specific membrane proteins.In addition to the resident macrophages of multiple tissues,CD68 is also expressed in TAMs.CD206,also called macrophage mannose receptor(MMR)or mannose receptor C type(MRC1),is a pattern recognition receptor with endocytosis and phagocytosis.CD206 is known to be expressed on most classes of macrophages and dendritic cell subpopulations,and is routinely used to identify the M2 phenotype.To date,both CD68 and CD206 have been the subjects of extensive structural and functional investigation in human diseases,especially in tumors.Ourstudy will explore the expression and significance of CD68 and CD206 in hepatocellular carcinoma(HCC)from three different levels of gene,histology and hematology.Objective: Ourproject has three parts.Part one: to explore the polymorphism of the three CD68 and CD206 SNPs(CD68 rs9901675?CD68rs9901673 and CD206 rs941)and their association with the risk,clinical characteristics and prognosis of HCC,to provide new breakthrough point for the research on the pathogenesis of HCC.Part two: to determine if the distribution and number of CD68-positive and CD206-positive macrophages changes and to evaluate the prognostic values in HCC post-operation patients.Part three: to explore the expression difference of solubleCD68 and CD206 in serum between HCC patients and health controls,diagnosis and prognosis potential,to provide a basis for individual clinical diagnosis and treatment and follow-up admiration of HCC.Methods:Part one: in this part,415 health controls and311 patients with primary HCC from the First Affiliated Hospital of Anhui Medical University were enrolled in this retrospective study between December 2014 and January 2017.Extraction of genomic DNA,detection and comparison of the genotype and allele frequencies in CD68SNPs(rs9901675 and rs9901673)and CD206rs941 locus between HCC patients and healthy controls have been conducted.Also,the associations of these SNPs with the risk,clinical characteristics and prognosis of HCC were analyzed.Part two: this part involving 268 patients with primary HCC who underwent hepatectomy was conducted betweenNovember 2011 andDecember 2014.CD68-positive and CD206-positivemacrophages infiltrating HCC tissues and adjacent tissues were detected by immunohistochemistry(IHC),and the relationships with clinicopathologic features and prognosis were addressed.Receiver operating characteristics(ROC)curve was used to calculate diagnostic accuracy.Overall survival(OS)and disease-free survival(DFS)were assessed in multivariate Cox models.Part three: 60 health controls,123 preoperative patients and 60 postoperative patients with primary HCCpatients from the First Affiliated Hospital of Anhui Medical University were enrolled in this retrospective study between February 2015 and January 2017.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of serum s CD68 and sCD206 and the association of them with clinical pathological features was analyzed.The diagnosis and prognosis value of serum sCD68 and sCD206 was investigated.Results:Part one: the SNP results demonstrated that CD68 rs9901675 polymorphism was associated with genetic susceptibility to HCC(OR = 0.409,95%CI0.206~0.8117,P = 0.009),there was statisticalsignificance in the three genotypefrequencies(GG,GA and AA)between HCC group and control group after adjusting for age and sex(P = 0.019)(96.46%?3.54% and 0% in the HCC group,92.53%?6.51% and 0.96% in the control group,respectively).However,the differences in allele frequencies of the other two SNPs(CD68 rs9901673,and CD206 rs941)were not statisticalsignificance(P> 0.05).Survival analysis showed that GA is an independent factor for HCCrelapse after surgery [HR(95%CI)= 8.114(1.103~59.675),P = 0.040].Nevertheless,there was no significant difference between CD68 rs9901675 polymorphism and clinical characteristics of HCC.Part two: first,infiltration increases in CD68 positive and CD206 positivemacrophage were observed in adjacent tumor tissues while less infiltration was found in tumor primary tissues(P< 0.05 andP< 0.05,respectively).HCC patients with portal hypertension(PHT)or different age,AFP level had different expression level of CD68 positive macrophages in tumor tissues,while there were different counts of CD68 positive macrophages in adjacent tumor tissues of cases with PHT,HBV positive,or different AFP level and tumor diameter(P < 0.05).As for CD206 positivemacrophages,HCC patients with different gender,Child-Pugh class had different expression in tumor tissues,whereas cases with cirrhosis,different total bilirubin level and TNM stage expressed variously in adjacent tissues(P< 0.05).Second,ROC curve showed that the optimum diagnostic cutoff for intratumor CD68 was 71/high-power fields(HPF)(area under curve(AUC)0.614,(95% CI 0.547-0.678),sensitivity 52.2% and specificity 70.7%,P = 0.003),and 87/HPF(AUC 0.599,(0.534-0.661),49.5% and 70.5%,P = 0.008)for adjacent tumoral CD68,compared with AFP above 400ng/ml.Moreover,the comparison of Kaplan-Meier survival curves showed that HCC patients with the increase of CD206 in tumor confers inferior OS and DFS(P = 0.014 and P = 0.003,respectively),while increased CD68 expression in adjacent tissues confers superior OS and DFS(P = 0.020 and P = 0.018,respectively).Cox regression analysis suggested both CD68 positive TAM density in adjacent tumor(P = 0.008)and intratumor CD206 positive TAM content(P = 0.028)as independent prognostic biomarkersfor postsurgical HCC patients.Furthermore,combination of CD68/CD206 and HBV-positive could further improve prognostic stratification,especially in DFS(P<0.05 and P< 0.05,respectively).Part three: the levels of serum sCD68 and sCD206 in preoperative or postoperative HCC patients were much higher than that in the health controls(P< 0.001).However,there was not difference of the levels of serum sCD68 and sCD206 between preoperative cases and postoperative ones(P >0.05).Meanwhile,there is correlation of the serum sCD68 or sCD206 levels between preoperative and postoperative cases(P< 0.05).Further analysis found that there were strong correlations between the levels of serum sCD68 and sCD206 and the clinical characteristics of HCC,including gender,HBV,liver cirrhosis,PHT and AFP.There was no significant difference in the serum levels of sCD68 and sCD206 among the genotypefrequencies of the three SNPs(CD68 rs9901675,CD68 rs9901673 and CD206 rs941)(P>0.05).ROC curve analysis demonstrated that the biggest area under the curve can been obtained when the levels ofpreoperative serum sCD68 was 207.46ng/ml and 20.52ng/ml for sCD206,and preoperativelevels of serum sCD206 reachedstatistical significance(P = 0.032)while sCD68 has no statistical significance(P = 0.174).Moreover,Survival analysis indicated thatthe overall survival time was much shorter in HCC patients with higher level of serum sCD68,PHT,lower level of albumin,lymphatic metastasis and distant metastasis(P< 0.05).Cox regression analysis suggested that the high level of serum sCD68 in postoperative HCC patients was an independent risk factorfor prognosis [HR(95%CI)=8.059(1.007~64.487),P = 0.049] while postoperative level of serum sCD206 and preoperative level of serum sCD206/ sCD68 had no significant significance(P> 0.05).Conclusions:These results suggest that CD68 rs9901675 polymorphism is associated with genetic susceptibility to HCC and the genotypefrequency of GG may be a risk factor for HCC pathogenesis and prognosis.Meanwhile,quantification of CD68/CD206 macrophages could be exploited to select HCC patients for multilayer postsurgical management and to provide the basis for novel strategies aimed at re-educating macrophages in the context of tumor microenvironment.Combining CD68/CD206 density with HBV-positive may further enhance the capability of HCC post-operative recurrence.Furthermore,Detection of level of serum sCD68/ sCD206 may contribute to observing the immune state of HCC patients,the preoperativelevels of serum sCD206 might be a biomarker for HCC early diagnosis and the high postoperativelevels of serum sCD68 could provide overall survival prediction for HCC patients.