Relationship Between Circulating Tumor Cells For Peripheral Blood And Postoperative Early Recurrence For Hepatocellular Carcinoma

Background:Primary liver cancer is a common malignant tumor of the digestive system.More than 50%of the patients live in China,and more than 90%of the primary liver cancer is hepatocellular carcinoma.Circulating tumor cells(CTCs)are a general term for various types of tumor cells present in the peripheral venous blood circulation.During tumor metastasis,cancer cells fall off from the primary tumor and enter the blood or lymphatic circulation system.Some of which have metastatic potential tumor cells survive in the circulatory system,and become circulating tumor cells,and develop into distant metastatic tumors.It has been confirmed that CTCs play a role in the metastasis of various tumors such as breast cancer,lung cancer,prostate cancer,and hepatocellular carcinoma.A large number of studies have shown that the numbers of CTCs in peripheral blood of hepatocellular carcinoma suggests that the stage is poor or the prognosis is poor.Peripheral venous blood CTCs represent the occurrence and development of primary to metastasis.The counting does not fully reflect the information contained in CTCs.It is necessary to further screen high-viability and high-metastatic CTCs subsets.The first generation of CTCs detection methods are based on the CellSearch(?)system of epithelial cell adhesion molecules,which is capable of detecting peripheral venous blood CTCs counts in most tumors(FDA approved for breast cancer,lung cancer,prostate cancer in 2004),however,It was found that during the process of distant metastasis of CTCs into the bloodstream,a process called epithelial-mesenchymal transition occurred.The peripheral blood CTCs of hepatocellular carcinoma were different from other epithelial tumors,and most of the liver cancer cells did not express Epithelial cell adhesion molecules,which limits the development of CellSearch(?)system technology in the detection of hepatocellular carcinoma.In 2017,the Ministry of Health of China issued a standard for diagnosis and treatment of primary liver cancer,pointing out that surgical treatment of hepatocellular carcinoma is the most important means for long-term survival of liver cancer patients.Liver resection is currently the main treatment in China,but hepatocellular carcinoma surgery 5 years after resection,the rate of tumor recurrence and metastasis is as high as 40%-70%.For high-risk recurrence,a large number of clinical studies have confirmed the effect of postoperative adjuvant transcatheter arterial chemoembolization.How to choose appropriate indications,although there are a small amount Retrospective case studies,but there are no corresponding guidelines and large-scale prospective controlled studies at home and abroad,and there is no corresponding model and test indicators for evaluating treatment effects.In this study,the CanPatrol TM system was used to evaluate the relationship between peripheral blood CTCs and prognosis in hepatocellular carcinoma.The system enriched CTCs by nanofiltration membrane method,and then identified and classified CTCs by multiplex probe RNA in situ hybridization.At the same time,the epithelial markers(EpCAM,CK8,CK18,CK19)and the interstitial markers(Twist,Vimentin)were detected to avoid the deficiency caused by the down-regulation or even non-expression of CTCs epithelial markers.Based on the CanPatrolTM system,we divided CTCs into epithelial,hybrid,and mesenchymal.We studied the relationship between CTCs typing and prognosis of hepatocellular carcinoma,and further analyzed the application of CTCs in the treatment of hepatocellular carcinoma after radical surgery.There is no literature report on this aspect of research.Purpose:The CanPatrol TM system was used to enrich and identify the CTCs of peripheral blood of hepatocellular carcinoma,calculate the positive rate of CTCs in this system,and evaluate the relationship between CTCs typing in peripheral blood of patients with hepatocellular carcinoma and early recurrence of hepatocellular carcinoma.The application of postoperative changes in the proportion of non-E-CTCs in peripheral venous blood in patients with cell carcinoma after hepatic cancer radical surgery.Method:All patients with hepatocellular carcinoma who underwent surgery from the Department of Hepatobiliary and Second Division of Zhujiang Hospital of Southern Medical University from October 2014 to April 2018 were selected as subjects.A total of 127 patients with hepatocellular carcinoma were enrolled in the study by retrospective case-control study.Of these,93 of them met the inclusion criteria.(1)Correlation between CTCs typing and early recurrenceAfter excluding patients who did not meet the requirements according to the exclusion criteria,we included 72 patients with hepatocellular carcinoma as subjects,and explored the relationship between peripheral blood CTCs typing and early recurrence.CTCs were detected in peripheral venous blood at 1 month postoperatively,and the positive rates of CTCs and total counts were calculated.According to the basis of the previous research of the Center,the early recurrence was defined as 6 months after operation(refer to the results of the study of wang z et al in 2018).The results were divided into Postoperative Early Recurrence(PER)and non-postoperative Recurrent group(non-PER).According to the median of the CTCs count in the PER group,we divided 72 subjects into high-count and low-count groups.The two groups were followed up for 2 years.The results were analyzed by K-M for single-factor survival analysis and Proportional Hazards Model was used for multivariate survival analysis to study the recurrence-free survival time between the two groups.(2)Application of CTCs in postoperative treatment monitoringAfter excluding patients who did not meet the requirements according to the exclusion criteria,we included 32 patients with hepatocellular carcinoma as subjects.All subjects underwent TACE treatment at 1 month and 2 months after surgery,respectively,before TACE treatment.At 3 months after surgery,peripheral venous blood was collected to detect CTCs and the proportion of non-E-CTCs was calculated.non-E-CTCs/CTCs detected were used as indicators to evaluate whether EMT occurred.If the ratios was greater than 0,it would be classified into EMT group,in contrast for non-EMT group,and the recurrence-free survival time between the two groups was compared.Results We used the K-M method for single factor survival analysis to compare the difference in recurrence-free survival between the two groups,and to evaluate the application of this index in postoperative adjuvant TACE therapy.Result:(1)Correlation between CTCs typing and early recurrenceWe examined peripheral venous blood in 72 patients with hepatocellular carcinoma(53 non-PER,19 with PER).CTCs were divided into epithelial,mixed,and interstitial CTCs.The positive rates of total counts of CTCs,epithelial CTCs,mixed CTCs,and interstitial CTCs were 90.3%,47.4%,73.7%,and 38.2%,respectively,compared with the traditional CellSearch(?)system positive rate(the reported positive rate was 60-80%in the literature).In addition,our previous study examined 21 patients with benign liver lesions with negative CTCs.We also studied 176 patients diagnosed of hepatic space-occupying lesions with B ultrasound examination.The results showed that the total CTCs count diagnosis was superior to AFP,and the using together could increase sensitivity.We calculated the median of the PER group,which was divided into a high-count group and a low-count group based on the median.We used the clinicopathological features,total CTCs,hybrid CTCs,and mesenchymal CTCs as influencing factors.Single-factor survival analysis of postoperative recurrence-free survival time showed that the tumor was large,tumors were multiple,vascular invasion,BCLC stage was later(B+C stage),mesenchymal CTCs were highly expressed and early recurrence of hepatocellular carcinoma patients is closely related.The above six factors were introduced into Proportional Hazards Model to show that the later BCLC stage(B+C stage)(HR=22.086(2.991-163.076);P=0.002)and a higher count of mesenchymal CTCs at 1 month after surgery(HR=2.243(1.048-4.798);P=0.037)was detected as an independent prognostic factor for early recurrence of hepatocellular carcinoma.(2)Application of CTCs in postoperative treatment monitoringPeripheral blood was successfully detected in 32 patients with hepatocellular carcinoma after radical operation.Among the PER groups,7 of 8 patients had a significant increase in non-E-CTCs counts.In the non-PER group,non-E-CTCs present reduced trend.According to the proportion of non-E-CTCs in peripheral venous blood CTCs,the patients were divided into EMT group(increasing proportion)and non-EMT group(inducing proportion).The K-M method were used to analyze the recurrence-free survival time between the two groups.The results showed that the median recurrence-free survival time in the EMT group was(5±2.357)months,the mean recurrence survival time was(6.375±1.435)months,and the non-EMT group was significantly prolonged.The Kaplan-Meier curves of the two groups were drawn and Log The-rank test showed statistically significant differences(x2 = 8.336,P =0.004).Conclusion:(1)We applied a new generation of CTCs detection methods.The results were divided into epithelial,Hybrid,and Mesenchymal CTCs.The positive rates of CTCs were 90.3%,47.4%,73.7%,and 38.2%,respectively,which were more positive rate than the traditional CellSearch(?)system.The rate(the positive rate reported in the literature is 60-80%)is significantly improved.(2)Peripheral venous blood mesenchymal CTCs in patients with hepatocellular carcinoma after 1 month were associated with early postoperative recurrence,but not epithelial CTCs.(3)Postoperative dynamic detection of non-E-CTCs/CTCs in patients with hepatocellular carcinoma can be used as a quantitative indicator for evaluating EMT.Postoperative dynamic testing can be used to evaluate the effectiveness of preventive treatment.