The Value Of Circulating Tumor Cell And Circulating Tumor DNA In The Efficacy Evaluation Of Transcatheter Arterial Chemoembolization For Hepatocellular Carcinoma

Part Ⅰ The predictive value of circulating tumor cell in the prognosis of patients with hepatocellular carcinoma treated with TACE Purpose:To assess the role of epithelial cell adhesion molecule(EpCAM)-positive circulating tumor cell(CTC)count in predicting survival outcomes of transcatheter arterial chemoembolization in patients with unresectable hepatocellular carcinoma(HCC).Materials and Methods:EpCAM-positive CTC counts were prospectively determined via CellSearch in peripheral blood of 97 patients with unresectable HCC treated with chemoembolization.The impact of each CTC cutoff point on overall survival(OS)was evaluated by univariate Cox regression analysis.Based on hazard ratio,patients were divided into 3 group with low(CTC count 0/1),moderate(CTC count 2-5),and high(CTC count>6)levels.Correlation of CTC counts with survival was assessed by Cox proportional-hazards model.Results:Eighty-nine patients met inclusion criteria and were enrolled.On multivariate Cox regression analysis,CTC count was found to be an independent predictor of OS(P=0.049)and progression-free survival(PFS;P=0.007)in patients treated with chemoembolization.After adjustment for confounding factors,mortality risks in the high-and moderate-level groups were 2.819 times(95%confidence interval[CI],1.218-6.526;P=0.016)and 1.301 times(95%CI,0.630-2.685;P=0.477)greater,respectively,than in the low-level group.The risk of progression was 3.705 fold higher in the high-level group(95%CI,1.628-8.433;P=0.002)and 1.648 fold higher in the moderate-level group(95%CI,0.843-3.223;P=0.144)vs the low-level group.Conclusion:High EpCAM-positive CTC count predicts poor survival of patients with unresectable HCC treated with chemoembolization.Part Ⅱ Effects of sorafenib on circulating tumor cell and survival prognosis in patients with hepatocellular carcinoma treated with TACE and its mechanism studyPurpose:To observe the effect of sorafenib on the number of circulating tumor cells and the survival prognosis of patients with hepatocellular carcinoma treated by TACE,and to observe the effect of sorafenib on the epithelial-mesenchymal Transition(EMT)of hepatocellular carcinoma cells through the experimental study.Materials and Methods:This retrospective analysis included all patients receiving either TACE plus sorafenib therapy or TACE alone for unresectable HCC between January 2014 and June 2019 at the First Affiliated Hospital of Soochow University.Overall 66 patients were enrolled into this study according to the inclusion and exclusion criteria.The primary outcome was overall survival(OS)and progression-free survival(PFS).A multivariate Cox proportional hazards analysis was conducted to examine determinants of OS,T-test was taken to compare the change of CTC number.At the same time,HepG2 cell lines were cultured in hypoxic culture to simulate local hypoxic microenvironment after TACE.CCK-8,Transwell and scratch tests were used to evaluate the effect of sorafenib on the proliferation,migration and invasion ability of HepG2 cells.The effects of sorafenib on the expression of E-cadherin,VE-cadherin and Vimentin in HepG2 cells were detected by Western-blot.The significance of the difference was compared by one-way analysis of variance.Results:The median OS of patients treated with TACE combined with sorafenib was significantly longer than those treated with TACE alone(16.3 vs.11.2 months;P=0.016),the median PFS was significantly longer than that of the TACE group(10 vs.4.5 months;P=0.001),The number of CTC in TACE combined with sorafenib group decreased significantly(2.66±0.6 vs.1.2±0.2).P=0.01),however,there was no significant decrease in the number of CTC in the TACE group after treatment(2.4±0.5 vs.1.6±0.3).P=0.084).The results of multi-factor analysis showed that treatment(P=0.003)and BCLC stage(P=0.006)were independent risk factors of overall survival of HCC patients.The proliferation,migration and invasion of HepG2 cells were enhanced after hypoxic culture,and changes in EMT-related protein expression were observed.Sorafenib can inhibit the proliferation,migration,invasion and EMT of HepG2 after hypoxia.Conclusion:Sorafenib can significantly reduce the amount of CTC in patients treated with TACE and significantly prolong the survival of patients.Sorafenib may reduce the production of CTC by inhibiting the epithelial mesenchymal transition of hepatocellular carcinoma cells.Part Ⅲ Preliminary study on the correlation between circulating tumor DNA and circulating tumor cell and the efficacy of TACE in the treatment of hepatocellular carcinoma patientsPurpose:To explore the value of circulating tumor DNA(ctDNA)in the short-term efficacy evaluation of TACE in patients with unresectable hepatocellular carcinoma,and the relationship between ctDNA and circulating tumor cell.Materials and Methods:Peripheral venous blood of patients with unresectable hepatocellular carcinoma before and after TACE treatment were collected,and the amount of CTC was detected.The plasma free DNA(cell-free DNA)was purified and extracted,and the ctDNA content of TP53 mutation was detected by real-time fluorescence quantitative PCR.Modified response evaluation criteria in solid tumors(mRECIST)was used to evaluate the postoperative clinical efficacy of patients treated with TACE,to compare the correlation between TP53 mutant ctDNA and CTC,as well as the objective response rate and disease control rate between the TP53 mutant group and the non-mutant group,and to analyze the correlation between ctDNA and the short-term efficacy of TACE.Results:A total of 30 patients with unresectable hepatocellular carcinoma were included in this study,of which 11 were positive for TP53 gene mutation,with a mutation rate of 36.7%.There were no statistically significant differences in age,gender,tumor size,number,BCLC stage,vascular invasion and extrahepatic metastasis between the TP53 mutant group and the non-mutant group(P>0.05).There was no significant difference in the amount of CTC between the TP53 mutant group and the non-mutant group(P>0.05).Six months after TACE,the objective response rate(ORR)and disease control rate(DCR)of patients in the TP53 mutant group were 12.5%and 25%,respectively,which were significantly lower than the 60%and 73.3%of patients in the non-mutant group,showing statistical differences(P=0.038 and P=0.037).Seven of the 11 patients in the mutant group underwent the second ctDNA test after TACE,showing that the changes in ctDNA content before and after TACE in 6 of the 7 patients were consistent with the short-term efficacy,with a consistency rate of 85.7%.Conclusion:There is no clear correlation between CTC and ctDNA of TP53 mutation,and TP53 mutation may be a risk factor affecting the short-term efficacy.The change trend of ctDNA content before and after TACE is consistent with the short-term efficacy,which is expected to be a reliable indicator to monitor the short-term efficacy of TACE.