| ObjectsTo find a suitable model of CTC-BIOPSY(?)platform used to detect circulating tumor cells of liver cancer;to study the significance of immunocytochemical double staining for qualitative detection of circulating tumor cells in liver cancer and to analyze the immunological and morphological characteristics of circulating tumor cells in liver cancer.To investigate the correlation between EPCAM+ CTCs and gender,age,liver cirrhosis,preoperative AFP,preoperative HBV,edmondson-steiner pathological grading,maximum tumor diameter,vascular thrombi,number of lesions and ki-67 proliferation index,as well as the correlation between clinical pathological characteristics and clinical prognosis.To analyze the guiding significance of the detection of EPCAM+ CTCs by immunocytochemistry double staining for the prognosis of patients.MethodsSixty-five patients with hepatocellular carcinoma confirmed by pathological examination in the department of hepatobiliary surgery,qian-fo-shan hospital,Shandong province,were selected as the study subjects.Preoperative peripheral blood,intraoperative hepatic venous blood and postoperative peripheral blood were collected.This study has been approved by the ethics committee of qian-fo-shan hospital in Shan dong province.65 subjects signed the informed consent before enrollment.The use of CTC-BIOPSY(?)(D100)and CTC-BIOPSY(?)(A10)platform,respectively evaluate the circulating tumor cells in the blood,and assess the dyeing effect,analysis of two kinds of model machine which is more suitable for detecting circulating tumor cells of liver cancer.The use of CTC-BIOPSY(?)(A10)sorting CTCs,using immunocytochemistry staining combined with morphological identification CTCs of liver cancer cells,to analyze the change of the patients with preoperative and postoperative CTCs and HCC CTCs morphological features;The clinical data of patients,such as gender,age,vascular thrombosis,etc.were collected by the case management system and the case workstation,and the correlation between various clinical factors and the number of EPCAM+ CTCs was analyzed.Results1.Select the detection platform:CTC-BIOPSY(?)(D100)detection rate of HCC for preoperative blood 8%(4/47)and intraoperative 12.7%(6/47),preoperative and intraoperative hepatic venous blood tube plugging rate is as high as 19.15%(9/47);under the microscope,the cells on the filter membrane were overlapped,with more cell membrane rupture,which was difficult to observe,and the staining color was deep,and the tumor cells were difficult to observe.Later we used CTC-BIOPSY(?)(A10)detect CTCs of liver cancer,the detection rate of preoperative and intraoperative blood CTCs was 54.34%(17/31)and 22.58%(7/3 1),preoperative and intraoperative hepatic venous blood blocking pipe did not occur.2.Choose test platform:CTC-BIOPSY(?)(D100)detection rate of HCC for preoperative 8%(4/47)and 12.7%(6/47),preoperative and intraoperative hepatic venous blood tube plugging rate is as high as 19.15%(9/47);Under the microscope,the cells on the filter membrane were overlapped,with more cell membrane rupture,which was diff-icult to observe,and the staining color was deep,and the tumor cells were difficult to observe.Later we choose CTC-BIOPSY(?)(A 10)detection(CTCs)for hepatocellular carcinoma(HCC),preoperative blood CTCs detection rate was 54.34%(17/31),intraoperative blood CTCs detection rate was 22.58%(7/31),preoperative and intraoperative hepatic venous blood blocking pipe did not occur.3.Preoperative and postoperative changes in the number of CTCs and the morphological and immunological characteristics of CTCs in hepatocellular carcinoma:By CTC-BIOPSY(?)(A 10)system,the morphological CTCs detection rate and the average CTCs count all showed postoperative(9.23%,on average 0.37)reduced compared with preoperative(32.3%,on average 2.43),the difference was statistically significant(P-0.0008);immunocytochemical staining showed that the CTCs granular membrane and cytoplasm were burnish(positive EPCAM and negative CD45),while the WBC showed burnish(positive CD45 and negative EPCAM).The positive rate of CTCs detected by immunocytochemical double staining method was 100%,and the coincidence rate between CTCs and morphology was 100%(21/21)before surgery,100%(13/13)during surgery,and 100%(6/6)after surgery.4.Relationship between EPCAM+ CTCs and clinical and pathological features:EPCAM+ CTCs was correlated with age and presence of intravascular thrombectomy in patients(P<0.05).However,it was not correlated with gender,liver cirrhosis,preoperative AFP,preoperative HBV,Edmondson-steiner pathological grading,maximum tumor diameter,number of lesions and ki-67 proliferation index.5.Relationship between clinic-pathological factors and prognosis of patients:Univariate analysis showed that liver cirrhosis,cancer thrombi,EPCAM+ CTCs,and Ki-67 proliferation indexes were statistically significant,which were significant independent prognostic factors affecting PFS(P<0.05).Multivariate analysis showed that EPCAM+ CTCs and cirrhosis were independent prognostic factor of PFS(P<0.05).6.Relationship between EPCAM+ CTCs and prognosis of patients:K-M curve analysis of the relationship between EPCAM+ CTCs and the survival and prognosis of patients with hepatocellular carcinoma.Log-Rank test indicated that the PFS of patients with EPCAM+ CTCs were significantly longer than those with EPCAM+ CTCs in peripheral blood,and the difference was statistically significant(P =0.015).Conclusion1.The CTC-BIOPSY(?)(A 10)is suitable for HCC detection of circulating tumor cells,solved the difficult problem CTCs separation technology of liver cancer.2.The method of immunocytochemical double staining combined with EPCAM and CD45 can effectively verify the CTCs of hepatocellular carcinoma,making a breakthrough in the qualitative study of CTCs of hepatocellular carcinoma.3.The majority of patients with hepatocellular carcinoma are middle-aged and elderly males,most of whom are associated with hepatitis or cirrhosis,and most of them are in the progressive stage when seeking treatment,which is consistent with the overall characteristics of China’s national conditions.EPCAM+ CTCs was correlated with age and existence of intravascular thrombectomy(P<0.05).4.Survival and prognosis of patients with liver cancer are affected by many clinical and pathological factors,with high heterogeneity.The existence of EPCAM+CTCs in peripheral blood is not only an independent prognostic factor but also a risk factor for PFS. |
