The Effect Of Anatomic Liver Resection On The Prognosis Of Hepatocellular Carcinoma With MVI And The Study Of Prognostic Markers

BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide,and its high mortality makes it the third leading cause of cancer death.Although in the past ten years,the research on the pathogenesis of HCC has continued to deepen,and various treatment methods such as immunity and targeted therapy have been continuously improved.The survival time of some HCC patients has been significantly prolonged.However,due to the high incidence of recurrence and metastasis,the prognosis of some HCC patients is still poor,the recurrence rate is still high(70%)5 years after radical treatment,and 15%of HCC patients develop extrahepatic metastases.An important mechanism of hepatocellular carcinoma metastasis is the ability of tumor cells to penetrate microvessels and spread to other sites through the bloodstream to form metastases.Studies have shown that HCC microvascular invasion(MVI)is one of the most important risk factors for HCC recurrence and metastasis after radical surgical resection.The domestic standard pathological diagnosis guideline for primary liver cancer defines MVI as:cancer cell nests are seen in the vascular lumen lined by endothelial cells under the microscope,which are more common in the small branches of the portal vein(including the blood vessels in the tumor capsule)in the adjacent liver tissue,which may be related to the portal vein as the main tumor vessel when hemodynamic disorders are present.Studies have shown that the incidence of MVI in HCC specimens ranges from 15%to 57%,and the presence of MVI increases the risk of recurrence after tumor resection and significantly shortens long-term survival.Another study showed that when MVI occurred in the distant adjacent tissue more than 1 cm from the tumor edge,the survival rate of HCC patients after radical hepatectomy was greatly reduced.Therefore,MVI is considered to be a decisive factor affecting the prognosis and early recurrence of HCC patients.Currently,liver resection is still one of the main treatment options for HCC patients with microvascular invasion,and safe surgical margins are a prerequisite for the complete removal of residual tiny tumor thrombi.With the development of surgical techniques and further understanding of liver anatomy,anatomical liver resection(ALR)is increasingly used in clinical practice.We defined ALR as complete resection of at least one segment of Couinaud containing the lesion and the portal vein in the drainage area of the lesion,whereas resection of tumors containing non-neoplastic liver parenchyma was considered non-anatomical liver resection(NALR).Compared with traditional non-anatomical resection(NALR),ALR can remove the vasculature in the tumor-bearing liver segment at the same time as the tumor is completely removed,which can reduce the occurrence of intraoperative bleeding,postoperative bile leakage and cross-sectional bleeding.rate and increase surgical safety.The results of our previous small cohort study showed that compared with non-anatomical hepatectomy,anatomical hepatectomy can improve the prognosis of patients with hepatocellular carcinoma,and anatomical hepatectomy can improve the prognosis of HCC patients with microvascular invasion.Due to the different anatomical location of microvascular infiltration in the liver,the impact of its MVI pathological stratification on anatomical hepatectomy remains unclear.Therefore,this study will retrospectively analyze the data of 198 patients who underwent radical hepatectomy and were diagnosed as HCC by postoperative pathology,focusing on the pathological grading of MVI,and exploring the clinical efficacy of anatomical hepatectomy in the treatment of HCC complicated with MVI.Targeted therapy is an emerging treatment modality based on effective biomarkers and a deeper understanding of tumor progression.In the present study,we assembled a cohort of HCC patients undergoing radical resection and further confirmed several potential biomarkers associated with microvascular invasion.Microfibrillar-associated protein 2(MFAP2),also known as microfibrillar-associated glycoprotein 1(MAGP1),interacts with microfibrillar proteins to regulate the function of microfibrils.It is the most widely distributed MAGP of most vertebrate protein and microfibrillar components.Previous studies have shown that the gene encoding MFAP2 is located at 1p36.13 and is associated with a variety of malignancies.Recent studies have also found that MFAP2 is overexpressed in hepatocellular carcinoma,but its role in the occurrence and development of hepatocellular carcinoma remains unclear.In this study,we further evaluated the role of MFAP2 in HCC by analyzing tumor specimens and detailed clinical data from HCC patients.Part Ⅰ Effect of anatomical liver resection on prognosis of patients with hepatocellular carcinoma with microvascular invasionObjectives:Microvascular invasion(MVI)of hepatocellular carcinoma(HCC)is considered to be a decisive factor affecting the metastasis and recurrence of tumor patients after radical therapy.Anatomical liver resection(ALR)is a radical treatment for HCC with liver segments as the basic resection unit.At the same time as the tumor,the vascular branches in the tumor liver segment were removed together.Studies have shown that compared with non-anatomical hepatectomy,anatomical hepatectomy can help improve the prognosis of HCC patients.Our previous small cohort study found that anatomical liver resection can improve the prognosis of HCC patients with MVI,but it is still unclear whether the pathological grade of MVI is related to anatomical liver resection.This section will focus on the pathological grading of MVI and discuss the effect of anatomical liver resection on the prognosis of patients with hepatocellular carcinoma(HCC)with microvascular invasion(MVI).Methods:First,the clinical data of patients undergoing radical hepatectomy for HCC in the Department of Liver Surgery of the First Affiliated Hospital of the University of Science and Technology of China from December 2009 to December 2019 were collected.According to the inclusion and exclusion criteria,a total of 569 cases met the inclusion criteria,and the general conditions,AJCC stages,surgical methods,and survival time of the included cases were sorted out.All the wax blocks of the cohort cases were obtained from the database of the Pathology Department of the First Affiliated Hospital of the University of Science and Technology of China.According to the MVI diagnostic criteria defined in the "Guidelines for Standardized Pathological Diagnosis of Primary Liver Cancer(2015 Edition)",the pathological grading of MVI was performed on all the enrolled cases by double-blind reading of the films by a chief physician and a deputy chief physician of the pathology department.All statistical analyses were performed using SPSS 22.0 software.The chi-square test or Fisher’s exact test was used for categorical variables,the survival curve was drawn by the Kaplan-Meier method,and the Log-rank method was used for the comparison between the two groups.P<0.05 was considered statistically significant.Findings:By performing PSM matching on 569 enrolled patients,165 and 164 patients were matched in the ALR and NALR groups,respectively.The results showed that compared with patients in the NALR group,patients who received ALR had longer overall survival and recurrence-free survival,and ALR could bring better clinical prognosis.198 patients screened for MVI pathological grading,111 underwent anatomical hepatectomy and 87 underwent non-anatomical hepatectomy.In MVI pathological staging,M0(no microvascular infiltration),M1(low microvascular infiltration),M2(high microvascular infiltration)group gender,alpha-fetoprotein(AFP)value,number of tumors and AJCC staging,and comparison of intraoperative and postoperative conditions No statistical significance(P>0.05).There were 35,37,and 39 patients undergoing anatomical hepatectomy in M0,M1,and M2 groups,respectively,and 20,31,and 36 patients underwent non-anatomical hepatectomy,respectively.There was no significant difference in the overall survival time between patients in the M0 group and the M1 group with or without anatomical hepatectomy(P>0.05).The median survival time of liver resection patients was 20 months,and the difference was statistically significant(P=0.003).Conclusion:Compared with non-anatomical hepatectomy,anatomical hepatectomy can significantly prolong the postoperative survival time of patients with hepatocellular carcinoma with MVI.For HCC patients with no microvascular invasion and low microvascular invasion in MVI pathological grades,anatomic liver resection had no significant effect on overall survival compared with non-anatomical liver resection.For advanced MVI,anatomic liver resection significantly improved survival in the cohort.Part Ⅱ Microfibril-associated protein 2(MFAP2)overexpression is closely associated with tumor angiogenesis,microvascular invasion and poor prognosis in hepatocellular carcinomaObjectives:In the previous part of the study,we found that for HCC patients with no and low microvascular invasion,anatomic liver resection had no significant effect on overall survival compared with non-anatomical liver resection.For high levels of MVI,anatomic liver resection significantly improved survival in the cohort.In the present study,we assembled a cohort of HCC patients undergoing radical resection and further confirmed several potential biomarkers associated with tumor angiogenesis and microvascular invasion.Among them,microfibrillar-associated protein 2(MFAP2),also known as microfibril-associated glycoprotein 1(MAGP1),interacts with microfibrillar proteins to regulate the function of microfibrils.Previous studies have shown that the gene encoding MFAP2 is located at 1p36.13 and is associated with a variety of malignancies.Recent studies have also found that MFAP2 is overexpressed in hepatocellular carcinoma,but its role in the occurrence and development of hepatocellular carcinoma remains unclear.In this study,we further evaluated the role of MFAP2 in HCC by analyzing tumor specimens and detailed clinical data from HCC patients.Method:In this study,we analyzed the expression pattern of MFAP2 in HCC using cohort data from The Cancer Genome Atlas(TCGA)database and GEPIA database.At the same time,we detected the expression of MFAP2 and epithelial-mesenchymal transition(EMT)-related proteins in pathological sections and fresh tissues of hepatocellular carcinoma.We have since then correlated MFAP2 expression with pathological characteristics of patients and identified risk factors associated with disease-free survival(DFS)and overall survival(OS).The role of MFAP2 in EMTinduced proliferation and migration of MHCC97H cells was investigated in vitro.We further investigated the effect of MFAP2 on vascular endothelial growth factor A(VEGFA)-induced tumor angiogenesis.Findings:Upregulation of MFAP2 expression was observed in hepatocellular carcinoma.This upregulation is often accompanied by aberrant expression of EMT-related marker proteins.In addition,analysis of clinical data from 94 patients with tumor tissue showed that MFAP2 expression was significantly positively correlated with low DFS and low postoperative OS.Through in vitro experiments,we show that silencing MFAP2 expression results in EMT inhibition,thereby inhibiting cell proliferation and migration.Furthermore,downregulation of MFAP2 inhibits tumor angiogenesis by inhibiting VEGFA.Conclusion:These findings suggest that MFAP2 is highly expressed in hepatocellular carcinoma with microvascular infiltration and has the potential to predict the prognosis of HCC patients.MFAP2 induces tumor proliferation and migration through EMT,and promotes tumor angiogenesis through VEGF-A,suggesting that MFAP2 may be a potential therapeutic target for HCC.Part Ⅲ MFAP2-LOX1 axis promotes angiogenesis and epithelialmesenchymal transition in hepatocellular carcinoma through PI3K/AKT/GSK3β pathwayObjectives:Through the study of the second part,we found that the expression of MFAP2 is associated with tumor invasion,metastasis and angiogenesis,and MFAP2 may be a prognostic biomarker in hepatocellular carcinoma(HCC).Further in vitro experiments found that MFAP2 silencing in tumors is usually accompanied by low expression of VEGFA and epithelial-mesenchymal transition(EMT),and the tumor cell culture supernatant that down-regulates MFAP2 expression has an inhibitory effect on endothelial cell tube formation.Therefore,we believe that MFAP2 may affect the progression of HCC by affecting the expression of VEGFA and EMT.To clarify how MFAP2 affects hilar cholangiocarcinoma progression.In this part,we further explored the potential mechanism of MFAP2 in promoting the progression of hepatocellular carcinoma.Method:In this study,we analyzed the expression pattern of the MFAP2-associated protein LOX-1 in HCC using cohort data from the Protein Interaction Database(String)database and the GEPIA database.At the same time,we detected the expression of LOX-1 and epithelial-mesenchymal transition(EMT)-related proteins in pathological sections and fresh tissues of hepatocellular carcinoma.The role of the MFAP2-LOX1 axis in epithelial-mesenchymal transition(EMT)-induced cell proliferation and migration was preliminarily investigated in vitro.We further investigated that the MFAP2-LOX1 axis promotes HCC angiogenesis and epithelialmesenchymal transition through the PI3K/AKT/GSK3β pathway.Findings:The protein association network of MFAP2 and VEG-a was predicted by the STRING database,and the results showed that LOX1,as the only key intermediate protein,connected MFAP2,VEGF,Vimentin and other proteins,and the high expression of LOX-1 in HCC significantly shortened the OS of liver cancer patients And the worse the TMN stage,the shorter the overall survival time of patients with high LOX-1 expression.The correlation between LOX-1 and MFAP2 mRNA expression levels was detected in fresh hepatocellular carcinoma tissues.Immunohistochemical staining showed that MFAP2 was significantly correlated with the immunohistochemical score of LOX-1 protein expression,and MFAP2 could induce the expression of LOX-1 in hepatocellular carcinoma by promoting the transcription of LOX-1.Analysis of CCK-8,scratch,and transwell experiments showed that LOX-1 plays an important role in the migration and invasion of MHCC97H cells.After silencing and overexpressing MFAP2 expression in MHCC97H cell line,the results showed that MFAP2 promotes migration and invasion and EMT in hepatocellular carcinoma by activating LOX-1 and then PI3K/Akt/GSK3β pathway.Conclusion:In this part,we explored the expression of MFAP2 and LOX1 in hepatocellular carcinoma for the first time,and discussed their clinical prognostic significance in HCC.We demonstrate that in HCC,the expression of MFAP2 and LOX-1 is a more sensitive prognostic biomarker for hepatocellular carcinoma than positive expression of either alone.Through in vitro experimental studies,we further confirmed the important role of LOX1 in MFAP2-induced HCC progression.This provides a certain promotion for the exploration of new targets for the treatment of hilar cholangiocarcinoma.The MFAP2-LOX1 axis and the tumor cell progression involved in the PI3K/AKT/GSK3β pathway may provide a theoretical basis for finding potential targets for HCC therapy...