A Clinical Study Of Preoperative Detection Of Circulating Tumor Cells Predict The Presence Of Microvascular Invasion In Hepatocellular Carcinoma Patients

?Background and Objectives?Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world.At present surgical resection and liver transplantation are recognized as the best treatment for HCC,but the high recurrence and mortality rate after surgery is still an important factor,which effect the prognosis of HCC.Vascular invasion of HCC is considered to be an aggressive biological behavior expressed by the tumor,which is the most important factor in predicting the recurrence of HCC patients.Vascular invasion of hepatocellular carcinoma can be divided into Macrovascular invasion and microvascular invasion(MVI).Vascular invasion is defined as a tumor that invades visible blood vessels,mainly involving portal and hepatic vein,and is now included in the Barcelona clinical liver cancer staging(BCLC)as part of it.For Macrovascular invasion can been diagnosed by imaging preoperatively.And MVI as a special intermediate stage of the development of HCC,through the invasion of portal vein or hepatic vein branch formation and cause intrahepatic recurrence or systemic metastasis,affecting the prognosis of patients.The definition of MVI as a microscopic visible small veins(central vein,portal vein or small vein outside the capsule)floating into the group of tumor cells and the surrounding the vascular endothelial cells around.However,the definition and classification of MVI have not been unified in the world.There is still a great heterogeneity in the evaluation of the tissue characteristics,prognosis and treatment of MVI.Therefore,the diagnosis of MVI is still dependent on postoperative pathological examination.Looking for preoperative prediction biomarker of MVI has become the hot poin of current research.Circulating tumor cells(CTCs)are rare tumor cells released from tumors into the blood stream that are thought to have a key role in cancer metastasis.Malignant tumors with poor prognosis are generally associated with early implantation of CTCs in the metastatic site,in which the liver tumor-derived cytokines or inflammatory stimuli produced by infection complications can be induced by upregulating cell adhesion molecules Hepatocellular carcinoma is attached to the portal vein and hepatic vein endothelial cells of the liver to form CTCs cell clusters under the action of platelet,tissue factor and various immune cells to promote the clonal proliferation of cancer cells,and then infiltrate the surrounding matrix and blood vessel wall,System cascade effect,and ultimately promote the formation of venous thrombus.In recent years,with the development of sensitive molecular technology,it is possible to separate and count peripheral blood CTCs.The results of this study show that the positive rate of CTCs and the number of CTCs are significantly correlated with tumor size,portal vein tumor thrombus,TNM staging and so on,and affect the prognosis of patients.But there is no relevant report on whether there is a correlation between CTCs and another special type of MVI that is violated by blood vessels.MVI as a special type of vascular invasion is HCC patients with postoperative recurrence of one of the independent risk factors,and liver cancer patients with postoperative recurrence there is a close relationship.At present,the clinical diagnosis of MVI still need to be postoperative histopathological examination,no specific cell markers can be effective in predicting the occurrence of MVI before surgery.In this study,we rely on Ficoll density gradient centrifugation combined with magnetic activated cell separation(MACS)to enrich hepatocellular carcinoma CTCs,and through CD45 immunomagnetic beads negative sorting.Marked lymphocytes in the blood,after sorting the magnetic field,the labeled CD45 lymphocytes were adsorbed on the sorting column,while the CTCs were collected by the column separation.The negative sorting method reduces the effect of tumor specificity on the enrichment of CTCs compared with the positive sorting method.In the selection of markers for the identification of CTCs in liver cancer,we have tried a number of indicators and found that the asialoglycoprotein receptor(ASGPR)and Carbamy phosphate synthetase 1(Carbamy phosphate synthetase 1),which specifically express hepatocarcinoma cells,,CPS1)in the identification of liver cancer CTCs help to improve the positive rate.After the preoperative trial to establish and verify the multicolor immunofluorescence staining method feasible after we collected liver cancer patients with peripheral blood CTCS before surgery and collected statistical data of clinical pathology,all patients through postoperative HE staining to determine whether the occurrence of MVI,the final combination of clinical pathology Data,single factor and multivariate analysis in order to explore the indicators related to MVI,and draw the receiver operating curve(ROC)test the independent factors and multi-factor combination of preoperative prediction of the clinical value of MVI The In order to improve the accuracy of preoperative diagnosis of MVI,for the further study of liver cancer for the CTCs and MVI individualized treatment provides a theoretical basis.?Methods?1.Establishment of the multicolor immunofluorescence ataining system in the detection of CTCs.The 5-ml peripheral blood of hepatocellular carcinoma was collected by EDTA tube.Following density gradient centrifugation with Ficoll-Paque PLUS(GE Healthcare Life Sciences,Little Chalfont,Buckinghamshire,United Kingdom),CTCs were enriched by extracting CD45-expressing leukocytes with magnetically labeled anti-CD45 monoclonal antibody(Miltenyi Biotec)according to the instructions.The remaining cells were cytocentrifuged on polylysine-coated slides.Slides were coincubated with a mouse monoclonal antibody cocktail against asialoglycoprotein receptor(ASGPR)and carbamoyl phosphate synthetase 1(CPS1)(Abcam,MA,USA)and a rat anti-human CD45 monoclonal antibody(Santa Cruz Biotechnology),followed by incubation with a Cy3-conjugated goat anti-mouse IgG antibody and an Alexa Fluor 488-conjugated rabbit anti-rat IgG antibody(Invitrogen,Carlsbad,CA,USA),and subsequent costaining with DAPI.Stained slides were assessed by fluorescence microscopy(IX71;Olympus,Tokyo,Japan),and images were captured from positive-stained and control specimens using the same detector sensitivity and exposure time.2.HE staining specimens were judged HCC MVI occurred:According to the "Guidelines for Pathological Diagnosis of Primary Hepatocellular Carcinoma(2015 Edition)",MVI is defined as a tiny vein(central vein,portal vein or small vein of the capsule)visible under the microscope.vascular endothelial cells were surrounded.All patients were screened by HE staining,through microscopic observation,to identify whether the occurrence of MVI.3.Statistical analysis of the correlation between CTCs and MVI:The clinical and pathological data of 108 patients with liver cancer were collected and analyzed,and CTCs was counted and counted.Continuous variables are expressed as mean plus or minus standard deviation.Categorical variables expressed as frequency and percentage,single factor analysis method using 2 test,multivariate analysis using Logistic regression analysis,and to determine the microvascular invasion independently related to the related factors,and use the ROC curve(Receiver Operating Characteristic Curve,ROC)clinical efficacy test and compare various factors.As a test standard,the P 0.05 was considered statistically significant,and all statistical analysis was performed with SPSS 18.0 software.?Results?1.ASGPR+CPS1/CD45 to identify indicators that can be used to separate,enrich the sample HCC CTCs,is a reliable and effective method,based on identification of CTCs multicolor immunofluorescence staining technique can be used to establish an accurate expression was determined HCC CTCs.In the 24 patients with HCC,the results showed that ASGPR+CPS1(+)/CD45(-)cells were detected in 18 patients with HCC,and the detection rate was 75%.12 cases of patients with other malignancies(two cases of esophageal cancer,2 cases of gastric cancer,gallbladder four cases,three cases of colon cancer and one case of breast cancer)and 10 healthy volunteers peripheral species,the remaining cancer patients and healthy volunteers undetected ASGPR+CPS1(+)/CD45(-)cells.2.The average number of CTCs in each 5ml blood of patients with HCC was 16±14,the detection rate was 47.2%.Postoperative pathology confirmed 44 cases of HCC patients was MVI(+),including 30 males and 14 females;64 patients with MVI(-),including 41 males and 23 females.64 cases of CTCs(+)patients,49 patients with serum AFP>400?g/L,44 patients with tumor diameter>5cm,51 cases with single tumor number and 57 cases with multiple tumors.Patients were divided into two groups(CTCs(+)and CTCs(-))based on the detection of CTCs in peripheral blood of patients with HCC.There were significant correlations between CTCs(+)and MVI expression,the numbers of tumor,DCP>40mAu/mL and prealbumin?170mg/L in peripheral blood,but there was no significant correlation with CTCs(+)and other pathological features.3.33 cases was detected CTCs(+)in 44 MVI(+)patients.The detection rate was 75%;18 cases was detected CTCs(+)in 64 MVI(-)patients,the detection rate was 28%.The positive rate of CTCs(+)in peripheral blood of patients was significantly higher than that of MVI(-)patients(P <0.001).4.The results showed that CTCs(+),AFP>400?g/L,DCP>40mAu/mL,tumor diameter and the number of tumor were related with MVI.Multivariate logistic regression analysis showed that CTCs(+),AFP>400?g/L,DCP>40mAu/mL and tumor diameter were independent risk factors for MVI.When the HCC patients serum DCP>40mAu/mL,AFP>400?g/L,the larger the tumor diameter,CTCs(+),the occurrence of MVI probability may higher.5.Multivariate analysis showed that the parameters of CTCs(+),AFP>400?g/L,DCP>40mAu/mL,and tumor diameter were independent predictors of MVI in HCC patients preoperatively(P= 0.001,0.020,0.002,0.001 respectively).The area under the curve(AUC)of receiver operating characteristic(ROC)of CTCs(+)was 0.73(specificity and sensitivity were 71.9% and 75%,respectively),CTCs(+)has better predictive value than AFP>400?g/L,DCP>40mAu/mL or tumor diameter.The area under the ROC curve of multi-factor combination was 0.87(specificity and sensitivity were 78.1% and 81.8%,respectively),which could beteer predict the MVI preoperatively.?Conclusion?Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.The detection of CTCs in peripheral blood of patients with HCC is closely related to MVI,which can predict the MVI and provide a theoretical basis for the further study of individual treatment in HCC patients.