EMT Phenotype Identification Of Peripheral CTCs In Diagnosis Of Primary Liver Cancer

Background and Objection:Primary liver cancer(PLC)is one of the most common malignancies in the world and has high morbidity and mortality.Nowadays surgical resection is the preferred treatment for most PLC patients.However,postoperative recurrence is the main factor that affects PLC prognosis.Currently,monitoring PLC recurrence mainly relies on ultrasonography,computed tomography(CT)examination and serum alpha fetoprotein(AFP)levels.However,these conventional monitoring methods cannot detect early recurrence in most cases.Therefore,it is crucial to identify sensitive biomarkers for the early diagnosis of PLC recurrence.Over the past decade,the detection of circulating tumor cells(CTCs)in the peripheral blood of cancer patients has gained more and more attention.Peripheral blood CTCs detection can serve as an ideal source for sampling because it is a simple,repeatable,and minimally invasive procedure.In fact,the invasive tumor cells originated from the primary tumor proliferate constantly;they become CTCs with invasion and metastasis potentials after leaving the tumor tissue and entering blood circulation.Recently,the function of CTCs in tumors diagnosis,recurrence,and metastasis has been under active investigation.In this study,we analyzed the difference of EMT phenotype CTCs between primary liver cancer and nonmalignant liver diseases,with an attempt to improve the accuracy of PLC diagnosis in clinical practice.However,studies on the relationship between CTCs subtypes and tumor recurrence have rarely been reported.In this study,peripheral CTCs subtypes were examined in patients with PLC,and their relationship with PLC recurrence was analyzed.The clinical value of detecting each subtype of CTCs for early prediction of PLC recurrence was also investigated.Methods and materialsThe first group,5-mL samples of peripheral blood were obtained from 73 observed patients after diagnosed with liver masses.And finally,45(median:55 range:18～77)were diagnosed as having primary liver cancer,and 13(median:50,range:35～74)were diagnosed as having nonmalignant liver diseases.The second group,62 primary liver cancer patients(58 males and 4 females,30-79 years old,with a median age of 55)who underwent radical resection at Zhujiang Hospital of Southern Medical University from March 2014 to March 2016 were enrolled in this prospective study.5-mL samples of peripheral blood were obtained from observed patients.We adopted the CanPatrolTM CTCs enrichment technique to detect EMT phenotype CTCs in peripheral blood.By using this technique,we detected CTCs for expression of four epithelial markers(cytokeratins8,18 and 19 and epithelial cell adhesion molecule)and two mesenchymal markers(Vimentin and Twist).Statistical analysis.All statistical calculations were performed with SPSS v21.0.P value<0.05 was considered statistically significant.Results:We applied CanPatrolTM System detected the EMT phenotype CTCs in peripheral blood as epithelial CTCs(stained for epithelial markers),mesenchymal CTCs(stained for mesenchymal markers),mixed CTCs(stained for epithelial markers and mesenchymal markers).Through the first research,we intend to evaluate the diagnostic value of EMT phenotype CTCs count in discrimination between primary liver cancer and nonmalignant liver diseases.Of 73 observed patients;3 patients did not undergo further diagnosis,10 patients were finally diagnosed as other malignant tumor liver metastasis,45 were diagnosed as having primary liver cancer,and 13 were diagnosed as having nonmalignant liver diseases.Furthermore,we intend to evaluate the diagnostic value of EMT phenotype CTCs count in discrimination between primary liver cancer and nonmalignant liver diseases.In the last research,Of the 62 PLC patients,26 were diagnosed with early recurrence(ER)and 36 did not experience recurrence.Comparison between the recurrence group and the non-recurrence group showed the total number of CTCs,mesenchymal CTCs and mixed CTCs in the recurrence group was significantly higher than in the non-recurrence group.Receiver operator characteristic(ROC)curve analysis was performed to define the positive cut-off values as followed:total number of CTCs ≥4,mesenchymal CTCs ≥1,and mixed CTCs ≥3.Analysis showed that portal vein tumor thrombus(hazard ratio[HR]= 2.905,P = 0.023)and mesenchymal CTCs positivity(HR = 3.453,P = 0.007)were independent risk factors for ER.The correlation between the presence of mesenchymal CTCs and time to recurrence was further examined,and the results showed significantly shortened postoperative disease-free survival in patients positive for mesenchymal CTCs(P<0.001).Conclusion(1).Our findings suggest that the EMT phenomenon is in CTCs from the blood of primary liver cancer.(2).EMT phenotype CTCs might be promising biomarkers for the diagnosis of primary liver cancer.(3).PLC patients with positive peripheral mesenchymal CTCs have more serious risk of ER,which could be a potential biomarker in PLC prognosis monitoring.(4).Mesenchymal CTCs and portal vein tumor thrombus were independent risk factors for early recurrence after PLC radical resection.Mesenchymal CTCs have more predictive strength than portal vein tumor thrombus with respect early recurrence outcomes.