| Objective:In this exploratory study,we aimed to investigate the clinical application value of CD90,epithelial cell ashesion molecule(EpCAM)and CD133 expression levels in peripheral blood in early diagnosis,clinical staging prediction,clinicopathological features and prognostic assessment in HCC patients.Methods:Sixty-five patients with primary liver cancer who were admitted to the Department of Hepatobiliary and Panabatic Surgery of the Third Affiliated Hospital of Kunming Medical University from October 2019 to January 2021 and had not received anti-tumor drugs therapy were selected as the study group,and 10 patients with benign liver tumor who were selected as the control group.Meanwhile,12 healthy subjects were selected as the negative control.Peripheral blood of 7.5ml was collected from the all subjects 1 day before treatment.Circulating tumor cells labeled with fluorescent antibodies CD90,EpCAM,and CD 133 were used to detecte by density gradient centrifugation and multi-parameter high-throughput flow cytometry(FCM)and the expression of stem cell markers CD90,EpCAM,and CD 133 were analyzed by combining with fluorescent signals and FSC/SSC.By comparing the expression levels of different phenotypes of CTC,Alpha fetoprotein(AFP),des-gamma-carboxy(DCP)and Alpha-L-fucosidase(AFU)to between the liver malignant tumor group and the liver benign tumor group.The expression levels of different phenotypes of CTCs and serological markers were studied for the diagnosis of hepatocellular carcinoma,the sensitivity,specificity and area under the AUC were calculated and the ROC curve was plotted.Chi-square test was used to analyze the relationship between CTCs expression levels of different phenotypes and clinicopathological characteristics of liver cancer patients.Patients were followed up and the Kaplan-Meier method was used to analyse the correlation evaluate the correlation between the expression of CD90,EpCAM and CD 133 and recurrence-free survival and metastasis-free survival.The t-test or non-parametric test was used to analyze the count data,and the chi-square test was used to analyze the relationship between the positive rates of CD90+CTC,EpCAM+CTC and CD133+CTC and the clinicopathological characteristics of liver cancer patients.Patients were followed up,and Kaplan-Meier method was used to draw the survival curve.Multivariate Cox risk regression model was used to analyze the prognostic factors of HCC patients.Results:1.Detection 1 CTC per 7.5 mL in peripheral blood was defined as CTC positive;conversely,it is defined as CTC negative.CD90+CTCs were detected in 48 cases(73.8%)of peripheral blood of 65 HCC patients,1 case(10%)of 10 benign liver tumors,but not in healthy subjects;EpCAM+CTCs were detected in 22 patients(33.8%),1 in 10 benign liver tumor patients(10.0%)and not in healthy subjects.CD133+CTCs were detected in 22 liver cancer patients(33.8%),4 in 10 benign liver tumors(40.0%)and not in healthy subjects.The expression levels of CD90+CTC,AFP and AFU in HCC patients were higher than those in benign liver tumor group,and the difference was significant(p<0.05);but there were no significant difference in EpCAM+CTCs and CD133+CTCs between the two groups(p>0.05).2.In this study,the diagnostic evaluation results of HCC showed that the sensitivity and specificity of CD90+CTC in the diagnosis of HCC was 66.15%,100.0%,AUROC was 0.852,95%CI=0.783～0.920,p<0.0001.The sensitivity and specificity of AFP in the diagnosis of HCC were 83.08%,100.0%,AUROC was 0.932,95%CI=0.875～0.990,p<0.0001.The sensitivity and specificity of CD90+CTC combined with AFP were 89.23%,100.0%,AUROC=0.977,95%CI=0.945-1.000,P<0.0001.The combined test is better than the single test in the diagnosis of hepatocellular carcinoma.3.Correlation analysis of clinicopathological factors between CTC and HCC patients:the expression of CD90+CTC was correlated with portal vein tumor embolism(χ2=4.879,p=0.027),patient ages(χ2=5.092,p=0.024),TNM stage(p=0.032),BCLC stage(χ2=7.088,p=0.029)was significantly correlated;the expression of CD90+CTC was most likely associated with disease progression(p=0.054).The expression of EpCAM+CTC was significantly correlated with disease progression in HCC patients(p=0.043).The expression of EpCAM+CTC is highly likely to be correlated with TNM(p=0.055).The expression of CD133+CTC was correlated with the history of alcohol consumption(χ2=6.414,p=0.011).4.The levels of T cell subsets(CD4+Th,CD8+Ts,Th/Ts,CD3+T),NK cells,IFN-y and cytokines(IL-10,IL-2,IL-4,IL-6,TNF-α)were not significantly different between CD90+CTC group and CD90-CTC group(p>0.05).5.In this study,univariate analysis of PFS showed that the progression-free survival of 65 HCC patients in EpCAM-CTC group was significantly longer than that in EpCAM+CTC group(13.627± 1.091 months vs.10.352± 1.968 months,p<0.05).PFS was longer in patients without portal thrombus than in patients with portal thrombus(13.419± 1.268 vs.10.788± 1.756,p<0.05).By multivariate analysis,EpCAM+CTC and portal embolism were found to be independent prognostic factors of HCC patients(p<0.05).Conclusions:CD90 and EpCAM can be used as potential HCC stem cell markers,providing scientific basis for the clinical application of circulating tumor stem cells in the diagnosis,treatment and prognosis evaluation of HCC. |
