| Background: Primary liver cancer ranks the second cause of death from malignant tumors in the Chinese population,90% of which are hepatocellular carcinoma(HCC)originating from liver cells.With the development of surgical technology,it is no longer a problem to completely remove the primary liver cancer and the visible vascular tumor thrombus.Surgical treatment of HCC with vascular tumor thrombus is an important manifestation of the "expansion of surgical indications" for patients with advanced HCC.However,with the expansion of surgical indications,the postoperative recurrence rate is bound to increase further,which is also a problem that cannot be ignored.HCC combined with large vessel invasion is still an independent risk factor affecting patients.However,there is still a certain proportion of patients with liver cancer with large vascular invasion,who have longer tumor-free survival and overall survival after surgery.Even if different patients in the same postoperative tumor-free survival period,due to the different recurrence patterns,the sensitivity of the MDT treatment mode after recurrence is different,and the overall survival period is still different.Therefore,standardized treatment of liver cancer with full consideration of individual factors is very important for patients with HCC and vascular.Part 1: Comprehensive analysis of clinicopathological characteristics affecting the prognosis of patients with resectable liver cancer combined with vascular tumor thrombus Purpose: Through comprehensive analysis of the prognosis,recurrence pattern and clinicopathological factors of patients with resectable HCC combined with vascular tumor thrombus,thus identify the benefiting groups of patients with HCC combined with vascular tumor thrombus undergoing surgical resection.Method: Retrospectively collected 196 patients who underwent "macro-radical hepatectomy(macro R0 resection)" at the department of hepatobiliary surgery,affiliated tumor hospital of guangxi medical university from January 2012 to May 2019 and were pathologically confirmed to be HCC.The Kaplan-Meier method was used to estimate the tumor-free survival curve(time to recurrence)and overall survival curve of 196 patients.Then,according to the different locations of recurrence,the recurrence patterns of 196 patients can be summarized into 4 categories: single intrahepatic recurrence,multiple intrahepatic recurrence,intrahepatic recurrence accompanied by large vessel invasion or extrahepatic metastasis,and simple extrahepatic metastasis recurrence.The Kaplan-Meier method was used to estimate the tumor-free survival time(relapse time)and the overall survival curve of patients with the four recurrence patterns,and the log-rank test was used to compare the differences in their survival curves.Then,univariate and multivariate COX model analysis was used to analyze the tumor-free survival and overall survival of 196 patients;The univariate and multivariate analysis of binary logistic regression analysis was used to analyze the influencing factors of postoperative recurrence patterns.Through the binary logistic regression analysis results to determine the "non-single recurrence pattern" risk factors to comprehensively construct a Nomogram prediction model,and finally plan to establish the "surgical benefit population" of HCC combined with vascular tumor thrombi.Result: 1、In this study,the median recurrence time of 196 HCC patients with vascular tumor thrombus was only 3 months(95%CI = 2.64–3.36);the median overall survival time was 13 months(95%CI = 9.8–16.33).2、196 HCC patients were classified into 5 categories according to the different types of vascular tumor thrombus: type I portal vein tumor thrombus(87 cases);type II portal vein tumor thrombus(63 cases);type III portal vein tumor thrombus(17 cases);Hepatic vein tumor thrombus(20 cases);Bile duct tumor thrombus(9 cases).The average RFS of patients with type III portal vein tumor thrombus was significantly lower than that of the other 4 types(all p<0.05);in addition,the average RFS of patients with hepatic vein tumor thrombus and bile duct tumor thrombus was also significantly higher than that of patients with type II portal vein tumor thrombus(all p<0.05),while the RFS difference between type I portal vein tumor thrombus and type II portal vein tumor thrombus is not statistically significant(p=0.313).Similarly,the average OS of patients with type III portal vein tumor thrombus is also significantly lower than other 4 types(all p<0.001);in addition,the average OS of patients with hepatic vein tumor thrombus was also significantly higher than that of patients with type II portal vein tumor thrombus(p=0.044),while type I portal vein tumor thrombus and type II portal vein tumor thrombus The difference in OS between patients with bile duct tumor thrombus and bile duct tumor thrombus is not statistically significant(all p>0.05).3、The postoperative recurrence pattern of 196 patients in the present study were summarized into 4 categories: single intrahepatic recurrence(16%);multiple intrahepatic recurrence(31%);intrahepatic recurrence with large vessel invasion or extrahepatic metastasis(44%);simple Extrahepatic metastasis(9%).The average recurrence time of patients with single intrahepatic recurrence was 18.1months(95%CI = 12.8–23.4 months),which was significantly higher than that of multiple intrahepatic recurrence models(6.4 months,95%CI = 4.3–8.5,P <0.001),extrahepatic metastasis and recurrence pattern(4.8 months,95%CI =3.1–6.4,P <0.001)and intrahepatic recurrence with vessel invasion or extrahepatic metastasis pattern(4.9 months,95%CI = 3.8–6.4,P <0.001);Similarly,the average overall survival time of patients with intrahepatic single recurrence model was 69.04 months(95%CI = 53.6-70.4 months),which was significantly higher than that of multiple intrahepatic recurrence models(17.3months,95 %CI = 13.6–21.0,P <0.001),extrahepatic metastasis and recurrence pattern(18.7 months,95%CI = 12.1–25.3,P <0.001),and intrahepatic recurrence accompanied by large vessel invasion or extrahepatic metastasis pattern(12.5 Month,95%CI = 10.4–14.6,p <0.001).4、The treatment of 196 patients in this study for recurrence can be divided into three categories: potential radical treatment,palliative treatment,and only symptomatic and supportive treatment.Among them,the vast majority of patients with single intrahepatic recurrence can obtain potential radical treatment(82.6%),and more than half of patients with multiple intrahepatic recurrences(61.7%)and patients with extrahepatic metastasis recurrence(54.7%)are palliative.But for most patients with intrahepatic recurrence accompanied by large vessel invasion or extrahepatic metastasis(63.6%),it is only symptomatic and supportive treatment.5 、 Multivariate COX model analysis of 196 patients showed that:alpha-fetoprotein(AFP)≥400ng/ml(p=0.033),tumor≥10cm(p=0.033),number of tumors >3(p=0.046),incomplete capsule(p=0.046),high Ki67-index index(p<0.001),multiple intrahepatic recurrence patterns(p<0.013),extrahepatic metastasis recurrence patterns(p<0.001)and recurrence classification Intrahepatic recurrence accompanied by large vessel invasion or extrahepatic metastasis patterns(p<0.001)are independent risk factors of RFS,while postoperative preventive TACE is an independent protective factor(p=0.002).Similarly,tumor≥10cm(p=0.039),tumor number >3(p=0.001),vascular tumor thrombus classification(type III portal vein tumor thrombus)(p=0.018),incomplete capsule(p=0.010),high Ki67-index index(p<0.001),postoperative complications(p=0.028),multiple intrahepatic recurrence patterns in recurrence classification(p<0.001),extrahepatic metastasis recurrence pattern(p <0.001),intrahepatic recurrence accompanied by large vessel invasion and extrahepatic metastasis pattern(p<0.001),and palliative treatment(p =0.029)and symptomatic treatment in the treatment after recurrence Supportive treatment(p<0.001)are all risk factors of RFS(all p<0.05),while postoperative preventive TACE is still an independent protective factor(p=0.002).6、Logistic regression model analysis showed: tumor≥10Cm(p =0.047),tumor number>3(p=0.026),incomplete envelope(p=0.045),vascular tumor thrombus classification(type III portal cancer Shuan)(p=0.048)and high Ki67-index index(p<0.001)are independent risk factors that lead to "non-single recurrence pattern".7 、 Integrate these 5 risk factors,postoperative nomogram prediction models based on "preoperative 4 factors" and "comprehensive 5 factors" were set up respectively.The C-index of the "preoperative 4 factors" model and "combined5 factors" model were 0.733 and 0.787,respectively.According to the Yoden-index,the optimal critical value predicted by the“preoperative 4-factor”model was 135.15,with the sensitivity was 57.9% and the specificity was 90.4%;the optimal critical value predicted by the“combined 5-factor” model was105.66,with the sensitivity 61.2% and the specificity87.1%.Conclusion1.The prognostic factors that affect HCC with vascular tumor thrombus have diverse characteristics,among which "recurrence time" and "recurrence pattern" are two important prognostic factors2.Tumor size(≥10cm),number of tumors(>3),incomplete capsule,type of tumor thrombus(type III portal vein tumor thrombus),and high Ki67 positive index are 5 common independent risk factors that affect the "recurrence time" and "recurrence pattern"3.The "preoperative 4 factors" and "comprehensive 5 factors" Nomogram models may initially realize a more accurate prediction of the "recurrence pattern" and the evaluation of the patient’s prognosis.However,its predictive power still needs to further expand the sample size and conduct multi-center prospective verification.Part 2: Investigation on the biological characteristics of HCC with vascular tumor thrombus with different prognosisPurpose: To explore the molecular biological characterization of HCC with vascular tumor thrombi with different prognosis,and screen out relevant markers to provide targets for subsequent intervention therapy.Method: The gene expression profiling was used to analyze the difference gene expression of HCC with vascular tumor thrombus with different prognosis;the candidate gene BCAT1 was screened,and its expression was verified in clinical samples by RT-PCR and immunohistochemistry;the construction of BCAT1-sh RNA was slow Virus interference vector,silenced the expression of BCAT1 in the cell line Hep G2,using CCK8 experiment,Annexin V combined with flow cytometry apoptosis experiment,Transwell migration/invasion experiment jointly explored the effect of BCAT1 silencing on the malignant potential of liver cancer cells.At the same time,to explore the regulation of BCAT1 on the "EMT pathway" of cancer cells.Result: 1、A total of 181 differential genes were identified in this study,of which 97 differential genes were significantly up-regulated(Fold change≥2),84 differential genes were significantly down-regulated(Fold change≤0.5);GO analysis shown that they were mainly involved Metabolic reprogramming pathways,cell proliferation processes,cellular immune-related pathways and biological processes such as cell adhesion and intercellular signal communication.In additions,a total of 11 signal pathways were enriched,including: PI3K-Akt signaling pathway,Chemical carcinogenesis,Glycolysis/Gluconeogenesis,Metabolism of xenobiotics by cytochrome P450,Intestinal immune network for Ig A production,Drug metabolism-cytochrome P450,beta-Alanine metabolism,Glycine,serine and threonine metabolism,Fatty acid degradation,Tyrosine metabolism.2、BCAT1 was significantly overexpressed in HCC tissues in the poor prognosis group(Mean fold change: 7.30;p<0.001).The expression verification of BCAT1 by RT-q PCR showed that the date CT value of BCAT1-m RNA in the poor prognosis group was 5.891±0.283,which was significantly lower than7.213±0.521 in the good prognosis group(p=0.014).In addition,the immunohistochemical results also indicated that the BCAT1 medium-strong positive rate(++~+++)in the poor prognosis group was 65.7%(23/35),which was significantly higher than the good prognosis group 38.7%(12/31)(p<0.05).3、When silencing the expression of BCAT1 in the liver cancer cell line Hep G2,it can significantly inhibit the proliferation of cancer cells.The anti-BCAT1 in vitro function test indicates that when silencing the expression of BCAT1 in the liver cancer cell line Hep G2,it can significantly inhibit the proliferation,anti-apoptosis,Invasion ability;with the silence of BCAT1,the expression of markers on the EMT pathway also reversed changes(E markers reversed up-regulation,M markers reversed down-regulation).Apoptosis and invasion ability;with the silence of BCAT1,the expression of markers on the EMT pathway also reversed changes(E markers reversed up-regulation,M markers reversed down-regulation).Conclusion1.High proliferative potential,metabolic reprogramming disorder and immunosuppression may be important molecular biological events leading to poor prognosis of patients with HCC with vascular tumor thrombi.2.The high expression of BCAT1 can promote the EMT pathway of liver cancer cancer cells to endow liver cancer cells with high malignant potential,which may be one of the reasons for the poor prognosis of patients. |
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