Objective: To explore a method for culturing hepatocellular carcinoma and tumor-infiltrating lymphocytes (HCC-TIL) and investigate the mechanism of TIL in killing tumors. Methods: The distribution of regulatory T cells (Treg) in HCC was detected by immunohistochemistry. Conventional TIL and oligoclonal TIL were isolated by the traditional method of enzyme digestion combined with mechanical treatment for whole HCC and micro HCC tissue block culturing method. MTT was used to compare the killing activity of TIL. Flow cytometry was used to analyze the proportion of CD8+ T cells and Treg cells in TIL. Tumor-bearing mice were established, and TIL adoptive immunotherapy was performed. Results: Treg cells were mainly distributed in the stroma of HCC. In vitro experiments showed oligoclonal TIL had higher cytotoxicity to tumor cells which negatively correlated with the proportion of Treg cells. In vivo experiments showed oligoclonal TIL had a higher anti-tumor effect. IFN-γ in peripheral blood and the positive rate of intratumoral lymphocytic infiltration in oligoclonal TIL group were both higher. TGF-β and IL-10 in peripheral blood and the positive rate of intratumoral FoxP3 and IL-17 were both lower than those in conventional TIL group. Conclusion: The oligoclonal TIL culture method could obtain TIL with higher purity, and cytotoxicity to tumor cells was associated with Treg cells. The oligoclonal TIL had cytotoxicity to autologous HCC cells and significant inhibitory effect on the growth of transplanted tumors. The mechanism might be associated with the inhibition of Treg cells proliferation, increase of IFN-γ secretion, and decrease of TGF-β, IL-10, and IL-17 secretion.

Circulating tumor cells (CTCs) are cancer cells shed from either the primary tumor or its metastases that circulate in the peripheral blood. The CTCs are regarded as the source of tumor recurrence and metastasis and speculated as the indicators of residual tumors, thereby indicating a poor prognosis. Although CTCs play a vital role in tumor metastasis and recurrence, little is known about the underlying survival mechanisms in the blood circulation. The accumulating evidence has revealed that CTCs might survive in the peripheral blood by overcoming the mechanical damage due to shear stress, resistance to anoikis, evasion of immune destruction, and resistance to chemotherapy. The present review addresses the putative survival mechanisms underlying the formation and migration of CTCs according to their biological characteristics and blood microenvironment. In addition, the relationship between CTCs and microenvironment is illustrated, and the influencing factors related to the interactions of CTCs with various components in the peripheral blood are reviewed with respect to the platelets, immune cells, cytokines, and circulating tumor microemboli (CTM). Furthermore, the recent advances in the new treatment strategies targeting the survival mechanisms of CTCs are also discussed.

Background: We intended to explore hepatitis B virus (HBV) reactivation after percutaneous radiofrequency ablation (PRFA) for HBV-related hepatocellular carcinoma (HCC) and the impact of antiviral therapy (AVT) on post-PRFA outcomes. Methods: Data on 538 consecutive patients who underwent PRFA for HBV-related early HCC at the Eastern Hepatobiliary Surgery Hospital between 2007 and 2011 were studied. Propensity score matching (PSM) analysis was used to compare the outcomes between the study groups. Recurrence free survival (RFS) and tumor recurrence were endpoints. Post-PRFA viral reactivation, hepatitis, and patterns of tumor recurrence were also observed. Logistic regression, Kaplan-Meier method and Cox proportional regression were used during the analysis. Results: Viral reactivation developed in 10.8% of patients who underwent PRFA. Patients with HBV reactivation had higher 1-, 3-, and 5-year tumor recurrence rates than patients without viral reactivation after PRFA (46.9%, 81.6% and 81.6% vs 36.0%, 63.5% and 65.4%, P = 0.004). AVT reduced viral reactivation rate (P < 0.001) and decreased 1-,3-,5-year tumor recurrence rate when compared with the no-AVT (30.2%, 58.6% and 61.4% vs 44.1%, 72.6% and 73.0%, P = 0.001). The local recurrence of tumor after PRFA was only associated with tumor diameter (P = 0.010), however, viral reactivation (P = 0.015) and AVT (P < 0.001) were independent risk factors of intrahepatic distant recurrence. Conclusions: HBV could be reactivated after PRFA. Viral reactivation and AVT had opposite impact on intrahepatic distant recurrence but not local tumor progression of HCC patients after PRFA. Legal entity responsible for the study: Feng Shen MD. Funding: State Key Project on Infectious Diseases of China (2012ZX10002-011,016 to FS). Disclosure: All authors have declared no conflicts of interest.

目的 研究精氨酸谷氨酸注射液对肝切除术后缺血再灌注损伤的保护作用及安全性.方法 选择2015年3月至2017年3于上海东方肝胆外科医院行择期肝部分切除患者93例,随机分为对照组和观察组,对照组46例、观察组47例.对照组予以常规保肝药,观察组予以精氨酸谷氨酸注射液治疗,比较两组黄嘌呤氧化酶(XOD)、热休克蛋白(HSP)、中性粒细胞、一氧化氮(NO)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、血氨、T细胞亚群及不良反应发生情况.结果 治疗后,观察组患者XOD、HSP、中性粒细胞下降趋势明显高于对照组(P＜0.05),观察组NO上升趋势优于对照组(P＜0.05);治疗后观察组患者AST、ALT及血氨下降程度均高于对照组(P＜0.05);治疗后,观察组T细胞亚群CD3+、CD4+、CD4+/CD8+下降幅度均高于对照组(P＜0.05),CD8+上升幅度高于对照组(P＜0.05);两组患者治疗期间的不良反应发生情况比较差异无统计学意义(P＞0.05).结论 精氨酸谷氨酸注射液能够减轻肝部分切除术后缺血再灌注损伤,保护肝功能,可能与其能够提高机体对氧自由基清除能力,抑制过氧化反应,改善机体免疫功能有关.

Partial hepatectomy is a potentially curative therapy for intrahepatic cholangiocarcinoma (ICC). Unfortunately, the overall surgical prognosis remains dismal and the actual 10-year survival has not been reported. This study aimed to document 10-year actual survival rates, identify the prognostic factors associated with 10-year survival rate, and analyze the characteristics of patients who survived ≥ 10 years. Among 251 patients who underwent curative liver resection for ICC between 2003 and 2006 at the Eastern Hepatobiliary Surgery Hospital, 21 patients (8.4%) survived ≥ 10 years. The 5-, 7-, and 10-year overall survival rates were 32.3%, 22.3% and 8.4%, respectively. The 10-year cumulative incidence of ICC-related death and recurrence were 80.9% and 85.7%, respectively. Multivariate analysis based on competing risk survival analysis identified that tumor > 5 cm was independently associated with ICC-related death and recurrence (hazard ratios: 1.369 and 1.445, respectively), in addition to carcinoembryonic antigen (CEA) >10 U/mL, carbohydrate antigen 19-9 (CA19-9) >39 U/mL, multiple nodules, vascular invasion, nodal metastasis and local extrahepatic invasion. Patients who survived ≥ 10 years had a longer time to first recurrence, lower levels of CEA, CA19-9 and alkaline phosphatase, less perioperative blood loss, solitary tumor, smaller tumor size, and absence of nodal metastasis or local extrahepatic invasion. In conclusion, a 10-year survival after liver resection for ICC is possible and can be expected in approximately 8.4% of patients.

Circulating tumor cells (CTCs) are a kind of tumor cells disseminated in the peripheral blood circulation due to spontaneous operation, diagnosis or treatment. CTCs have been regarded as crucial source of tumor recurrence and metastasis. The recent studies show that CTCs can survive in circulatory system through overcoming mechanical damage of blood shear stress, evading immune destruction, and resistance to anoikis and systemic chemotherapy drugs. In this paper, the possible survival mechanisms involved in CTCs formation and migration are reviewed according to their biological characteristics and blood microenvironment, while the latest progress in the novel strategies for tumor treatment targeting CTCs is discussed. DOI:10.3781/j.issn.1000-7431.2017.55.007

Objective To investigate the application value of hepatic portal reocclusion for the prevention of bile leakage after hepatectomy. Methods In this prospective study, 197 patients who underwent hepatectomy alone in the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University between March 2014 and November 2014 were recruited. According to the random number talbe method, the patients were divided into the hepatic portal reocclusion group (n=99) and traditional surgery group (n=98). In the hepatic portal reocclusion group, 81 cases were males and 18 females, aged (54±11) years old on average. Among them, 89 cases were diagnosed with primary liver cancer and 10 with benign liver lesions. After the liver resection with Pringle maneuver, the first porta hepatis was reoccluded to elevate the pressure of intrahepatic blie duct, and the bile duct was examined and tightly sutured. In the traditional surgery group, 82 cases were males and 16 females, aged (52±10) years old on average. Among them, 91 cases were diagnosed with primary liver cancer and 7 with benign liver lesions. The liver resection was performed using Pringle maneuver. The informed consents of all patients were obtained and the local ethical committee approval was received. The perioperative status and prognosis were observed and compared between two groups. The hepatic portal occlusion time and operation time between two groups were compared using t test or Kruskal-Wallis rank test. The rates were compared using Chi-square test or Fisher's exact probability test. Results The frequency of hepatic portal occlusion in the hepatic portal reocclusion group was 2(1-4), significantly higher than 1(1-3) in the traditional surgery group (Z=0.000, P<0.05). The hepatic portal occlusion time in the hepatic portal reocclusion group was (21±10) min, significantly longer than (17±9) min in the traditional surgery group (t=0.001, P<0.05). The postoperative length of hospital stay in the hepatic portal reocclusion group was (8±3) d, significantly shorter than (9±3) d in the traditional surgery group (t=-0.040, P<0.05). The incidence of postoperative bile leakage in the hepatic portal reocclusion group was 1%(1/99), significantly lower than 9%(9/98) in the traditional surgery group (χ2=6.830, P<0.05). The symptoms of bile leakage were effectively controlled after short-term drainage. Conclusions Application of hepatic portal reocclusion can effectively reduce the incidence of bile leakage after hepatectomy, and provides a simple and efficacious approach to prevent the incidence of bile leakage for the surgeons. Key words: Hepatectomy; Biliary fistula; Hepatic portal reocclusion

Midkine is overexpressed in hepatocellular carcinoma (HCC) and plays a role in tumor progression, but less is known about its role in resistance of circulating tumor cells (CTCs) to anoikis which leading to recurrence and metastasis. The aim of the present study was to analyze whether midkine was associated with HCC progression with anoikis resistance. We found that cultured HCC cells were more resistant to anoikis, which paralleled midkine expression, and midkine treatment significantly inhibited anoikis in a dose-dependent manner. Furthermore, in in vitro and in vivo assays, knockdown of midkine resulted in significant sensitivity to anoikis, decreased cell survival and significantly decreased tumor occurrence rate. Patients with midkine-elevated HCC had higher CTC counts and less apoptotic CTCs, as well as significantly higher recurrence rate and shorter recurrence-free interval. To understand the molecular mechanism underlying the midkine with HCC progression, we performed in vitro and in vivo studies. We found that midkine plays an important role in enhancement of HCC cell resistance to anoikis, thereby promoting subsequent metastasis. Activation of PI3K/Akt/NF-κB/TrkB signaling by midkine-activated anaplastic lymphomakinase (ALK) is responsible for anoikis resistance.

As negative immune regulatory molecules,the inhibitory co-stimulatory molecules programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) play important roles in the adaptive cellular immunity.PD-L1 expressed in tumor cells is involved in regulating T cell activation and differentiation and inhibiting the anti-tumor immune activity of T cell through specific binding with the receptor molecule PD-1 on T cells.Currently,a variety of agents targeting the immune checkpoints of PD-t/PD-L1 have been used in clinic,which have exhibited long-lasting effect in treatment for different types of tumors.In this review,we summed up the molecular structure,expression features,factors influencing upregulation of PD-1/PD-L1,and their roles in promoting tumor growth and escaping from immune system and tumor immunotherapy.

BACKGROUND/AIMS:To explore the possibility and feasibility of hepatic portal reocclusion for detecting bile leakage during hepatectomy.METHODS:Data were prospectively collected from 200 patients who underwent hepatectomy alone for removal of various benign or malignant tumors between March 2014 and November 2014. The surgical procedure used a conventional method for all patients, and one additional step (hepatic portal reocclusion) was included in group B. The postoperative outcomes of the patients in group A (subjected to the traditional procedure) and group B (subjected to hepatic portal reocclusion) were compared during the same period, and the incidence rates of postoperative bile leakage and other complications in the 2 groups were also analyzed.RESULTS:The incidence of postoperative bile leakage in group B was significantly lower than that in group A (1.0 vs. 9.2%, p = 0.009), although no significant differences in postoperative indicators of liver dysfunction and other complications were observed between the 2 groups (p > 0.05).CONCLUSIONS:Hepatic portal reocclusion effectively reduced the incidence of bile leakage compared to the traditional procedure, without significantly affecting liver function. Therefore, this method might be an alternative to other tests for bile leakage.

目的 探讨术前外周血循环肿瘤细胞(CTCs)检测对肝细胞肝癌(HCC)微血管侵犯(MVI)的预测价值.方法 收集首次手术切除治疗的HCC患者108例,术前未行抗肿瘤治疗;术前取外周静脉血检测CTCs,术中取病理HE染色判断是否发生MVI;收集患者临床资料,进行发生MVI的单因素及多因素分析,以受试者工作曲线(ROC)判断各独立因素术前预测肝癌MVI的价值.结果 108例肝癌患者术前外周血检出CTC 51例,术后病理证实发生MVI 44例,且发生MVI者外周血检CTC 阳性检出率高于未发生MVI者(P<0.05).多因素分析显示,CTC阳性(OR=5.218,95%CI 1.700~16.011,P=0.004)、AFP>400 μg/L(OR=4.803,95%CI 1.714~13.568,P=0.003)、DCP>40 mAU/mL(OR=3.046,95%CI 1.055~8.797,P=0.040)和肿瘤直径(OR=4.332,95%CI 1.544~12.1521,P=0.005)是MVI发生的独立危险因素.ROC 分析显示,CTC阳性的曲线下面积及灵敏性、特异性均高于其他3种危险因素.结论 HCC患者术前外周血CTC检测能较好地预测MVI的发生,可为后续个体化治疗提供参考.

Background. Tumor recurrence after liver resection for intrahepatic cholangiocarcinoma is common. The effective treatment for recurrent intrahepatic cholangiocarcinoma remains to be established. This study evaluated the short- and long-term prognoses of patients after repeat hepatic resection for recurrent intrahepatic cholangiocarcinoma.Methods. Data for 72 patients who underwent RO repeat hepatic resection for recurrent intrahepatic cholangiocarcinoma at the Eastern Hepatobiliary Surgery Hospital between 2005 and 2013 were analyzed. "Tumor re-recurrence, recurrence-to-death survival, and overall survival were calculated and compared using the Kaplan-Meier method and the log-rank test. Independent risk factors were identified by Cox regression analysis.Results. Operative morbidity and mortality rates were 18.1 % and 1.4%, respectively. The 1-, 2-, and 3-year re-recurrence rates were 53.2%, 80.2%, and 92.6%, respectively, and the corresponding recurrence-to-death survival was 82.9%, 53.0%, and 35.3 %, respectively. The 1-, 3-, and 5-year overall survival was 97.2%, 67.0%, and 41.9%, respectively. Patients with a time to recurrence of > 1 year from the initial hepatectomy achieved higher 1-, 2-, and 3-year recurrence-to-death survival than patients with a time to recurrence of <= 1 year (92.5 %, 61.7%. and 46.6% vs 70.4%, 42.2%, and 23.0%, P = .022). Multivariate analysis identified that recurrent tumor > 3 cm (hazard ratio: 2.346; 95 % confidence interval: 1.288-4.274), multiple recurrent nodules (2.304; 1.049-5.059), cirrhosis (3.165; 1.543-6.491), and a time to recurrence of <= 1 year (1.872; 1.055-3.324) were independent risk factors of recurrence-to-death survival.Conclusion. Repeat hepatic resection for recurrent intrahepatic cholangiocarcinoma was safe and produced long-term survival outcomes in selected patients based on prognostic stratification with the presence of the independent risk factors of recurrence-to-death survival.

BACKGROUND:Splenosis is a benign and relatively uncommon condition caused by trauma or splenectomy or other procedures involving splenic tissue. It is usually asymptomatic, and often diagnosed accidentally, especially misdiagnosed as malignant tumor.METHODS:A 54-year-old man with prior history of chronic hepatitis B virus infection and underwent splenectomy for traumatic splenic rupture following a traffic accident 23 years previously was admitted to our hospital and found a hepatic mass in the right upper quadrant during an imaging examination. The diagnosis of his was not clear and finally he agreed to receive a surgical treatment.RESULTS:During the operation, we found a mass in the right posterior lobe of the liver and a hard nodule on the right side of the diaphragm, both were completely resected, and postoperative histopathologic examination revealed that all excised tissues were proved to have histological structure typical for the spleen.CONCLUSIONS:The occurrence of intrahepatic splenosis is rare with only few cases previously reported in the literature. It is a benign disease and sometimes difficult to distinguish from diseases of the liver. The need for positive surgical resection of splenosis is still controversial.

肠道菌群在维持人体内环境稳态方面起着至关重要的作用,其与疾病的关系越来越多地受到重视,但相关病理生理学机制还远不清楚.近期的研究证实,肠道菌群失调参与肝癌的发生和发展过程,涉及由菌群失调引起的胆汁酸代谢改变、肝星状细胞衰老、内毒素代谢紊乱等.本文就肠道菌群失调诱导的这些变化促进肝癌发生和发展的作用机制研究进展作一综述,指出靶向肠道菌群失调的肝癌预防策略和未来研究方向.

The relationship between serum carcinoembryonic antigen (CEA) and postoperative prognosis in hepatocellular carcinoma (HCC) has not been reported.

Our aim in this study was to develop a prognostic scoring system with which to identify patients most likely to benefit from adjuvant chemolipiodolization (ACL) after liver resection for hepatocellular carcinoma (HCC). Data from 1150 HCC patients who underwent liver resection between 2002 and 2008 at the Eastern Hepatobiliary Surgery Hospital were used to develop the scoring system. Patients were stratified into prognostic subgroups using the new scoring system, and the outcomes of patients who received ACL and those who did not were compared in each subgroup. Using data from 379 patients operated on between 2008 and 2010 for validation, the scoring system had a concordance index (C-index) of 0.75 for predicting post-resectional overall survival (OS). It optimally stratified patients into three prognostic subgroups with scores of 0-5, 6-9 and ≥ 10, having better, medium and worse survival outcomes, respectively. A difference in OS between ACL and non-ACL patients was only detected in the subgroup with scores ≥ 10 (1-, 3-, and 5-year OS rates: 63.9%, 22.6%, and 9.0% vs. 33.8%, 5.6%, and 2.8%, p = 0.001). Our proposed scoring system provides an effective tool for selecting the patients most likely to benefit from ACL.

Repeat hepatectomy (re-hepatectomy) is an effective treatment for patients with intrahepatic recurrence following liver resection for hepatocellular carcinoma (HCC).

BackgroundAxl is a receptor tyrosine kinase which plays an important role in multiple human malignancies.DesignThe Axl expression was examined in several hepatocellular carcinoma(HCC) cell lines, paired tumor and nontumorous samples. Then, we examined cell growth curve, cell apoptosis and cell migration in SMMC-7721 cells over-expressed with Axl or siRNA against Axl, respectively. Finally, the prognostic value of Axl was investigated in a prospective cohort of 246 consecutive HCC patients undergoing curative hepatoectomy.ResultsWe found Axl was positive in 22% of examined tumor tissues and all four cell lines. Over-expressing Axl in SMMC-7721 cells accelerated cell growth, cell migration and inhibited cell apoptosis, while knock-down of Axl exerted opposite effect. Axl expression was closely associated with serum AFP, multiple tumors, absence of encapsulation, microvascular invasion, and advanced BCLC or TNM stage. Patients with positive Axl staining had a higher 5-year recurrence rate (92% vs. 71%, P<0.001) and a lower 5-year survival rate (9% vs. 48%, P<0.001) than those with negative staining. The multivariate analyses showed that Axl expression was an independent factor for both tumor recurrence (HR: 1.725; 95% CI: 1.219-2.441) and survival (1.847; 1.291-2.642).Conclusion Axl expression suggests more aggressive tumor invasiveness and predicts worse prognosis for HCC patients undergoing resection.

Objective To investigate the long-term outcomes of liver resection in the treatment of 1 370 patients with intrahepatic cholangiocarcinoma (ICC) and the related factors affecting tumor recurrence and patients' prognosis.Methods The retrospective cohort study was adopted.The clinicopathological data of 1 370 patients with ICC who underwent liver resection at the Eastern Hepatobiliary Surgery Hospital between January 2005 and December 2012 were collected.Patients received laboratory and imaging examinations,and then surgical plan was determined according to the preoperative results.Observation indicators included (1) preoperative examinations results:liver function,tumor markers and imaging examination,(2) surgical treatment:surgical procedures,operation time,volume of intraoperative blood loss,intraoperative blood transfusion,hepatic inflow occlusion,postoperative complications and duration of hospital stay,(3) postoperative pathological examination:tumor differentiation,vascular invasion,lymph node metastasis,local invasion and TNM stage,(4) results of follow-up:tumor metastasis and postoperative survival.(5) There were univariate analysis and multivariate analysis affecting postoperative tumor early recurrence.(6) There were univariate analysis and multivariate analysis affecting postoperative patients' prognosis.(7) Patients' survival risk was stratified to 3 subgroups,namely,low score group,median score group and high score group,based on tertiles of their nomogram scores.The follow-up using outpatient examination,telephone interview and letters was performed once every 2-3 months within 2 years postoperatively and once every 3-6 months after 2 years postoperatively up to November 15,2014.The follow-up included that data collection of medical history and physical examination,levels of CA19-9,carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP),liver function,routine blood test,chest X-ray and abdominal ultrasound.The above examinations were applied to patients in advance who were confirmed as suspected recurrence.Diagnosis of recurrence depended on results of imaging examination and clinical manifestations.Continuous variables were represented as M (range).Normality test was done using the ShapiroWilk test,and comparisons among groups with non-normal distribution and normal distribution were analyzed by the Mann-Whitney U test and t test,respectively.Ctegorical variables were analyzed using the chi-square test or Fisher's exact test.Postoperative recurrence rate and overall survival of patients were calculated by the life table method.The survival curve was drawn by the Kaplan-Meier method,and the survival rate was analyzed using the Log-rank test.The univariate analysis and multivariate analysis were done using the COX regression model.The concordance index (c-index) of survival probability was predicted by the nomogram and calibration curve was done by the R version 2.14.1.Results (1) Results of preoperative examinations in 1370 patients with ICC:liver function:levels of glutamyltranspeptidase (GGT),total bilirubin (TBil),glutamic-pyruvic transaminase (GPT),albumin (Alb) and platelet (PLT) were 71.9 U/L (range,40.0-162.0 U/L),12.7 μmol/L (range,9.7-17.2 μmol/L),27.4 U/L(range,18.0-45.0 U/L),42.2 g/L(range,39.5-44.9 g/L) and 191 × 109/L[range,(145-237) × 109/L],respectively.Tumor markers:levels of AFP,CA19-9 and CEA were respectively 3.8 μg/L (range,2.3-9.5 μg/L),44.2 U/mL(range,16.3 U/mL-257.6 U/mL) and 2.7 μg/L(range,1.6-5.3 μg/L).Results of imaging examination:tumor diameter was 5.7 cm(range,4.0-8.0 cm).There were 978 patients with solitary tumors,392 with multiple tumors,1 069 without liver cirrhosis and 301 with liver cirrhosis.(2) Surgical treatment:all the 1370 patients with ICC underwent liver resection,including 454 patients with number of hepatic segments resected ≥3 and 916 with number of hepatic segments resected ＜ 3.Operation time,volume of intraoperative blood loss,number of patients with blood transfusion and complications and duration of hospital stay were 120.0 minutes (range,60.0-280.0 minutes),300 mL (range,50.0-8 000.0 mL),261,408 and 16.0 days (range,13.0-20.0days),respectively.There were 411 patients with hepatic flow occlusion time ＞ 20 minutes and 959 patients with hepatic flow occlusion time ＜ 20 minutes.(3) Postoperative pathological examination:of 1370 patients with ICC,32,1 198 and 140 patients were respectively detected in the high-,moderate-and low-differentiated tumors,203,270 and 97 patients had respectively vascular invasion,lymph node metastasis and local invasion,and 706,327,57 and 280 patients were detected in stage Ⅰ,Ⅱ,Ⅲ and Ⅳ of TNM stage.(4) Results of follow-up:1 359 were followed up for a median time of 23.5 months (range,1.0-103.3 months).A median tumor recurrence time,1-,3-and 5-year recurrence rates were 16.2 months (range,13.9-18.5 months),42.8％,67.5％ and 75.4％,respectively.There were 555 patients with early recurrence.A median survival time,1-,3-and 5-year overall survival rates were 26.1 months(range,23.4-28.7 months),68.0％,41.9％ and 32.5％,respectively.(5) Results of univariate analysis showed that GGT,AFP,CA19-9,CEA,tumor diameter,number of tumors,method of liver resection,intraoperative blood transfusion,vascular invasion,lymph node metastasis and local invasion were risk factors affecting tumor early recurrence after liver resection of ICC [HR =1.313,1.217,1.352,1.346,1.476,1.928,1.241,1.295,2.180,2.152,2.119,95％ confidence interval (CI):1.133-1.521,1.013-1.461,1.167-1.567,1.109-1.633,1.271-1.715,1.656-2.245,1.067-1.445,1.084-1.547,1.815-2.619,1.826-2.536,1.655-2.715,P ＜0.05].Results of multivariate analysis showed that tumor diameter ＞ 5 cm,multiple tumors,vascular invasion,lymph node metastasis and local invasion were independent risk factors affecting tumor early recurrence after liver resection of ICC (HR =1.830,1.598,1.693,1.773,1.539,95％ CI:1.098-1.497,1.364-1.873,1.395-2.054,1.437-2.041,1.190-1.989,P ＜0.05).(6) Results of univariate analysis showed that GGT,Alb,CA19-9,CEA,tumor diameter,number of tumors,method of liver resection,intraoperative blood transfusion,vascular invasion,lymph node metastasis and local invasion were risk factors affecting patients' prognosis after liver resection of ICC (HR=1.401,1.496,1.759,1.759,1.588,1.947,1.284,1.356,2.052,2.513,2.357,95％ CI:1.213-1.618,1.144-1.955,1.519-2.036,1.469-2.106,1.371-1.840,1.679-2.257,1.107-1.489,1.143-1.608,1.718-2.452,2.142-2.948,1.859-2.988,P ＜ 0.05).Results of multivariate analysis showed that CA19-9 ＞ 39 U/mL,CEA ＞ 10 μg/mL,tumor diameter ＞ 5 cm,multiple tumors,vascular invasion,lymph node metastasis and local invasion were independent risk factors affecting patients' prognosis after liver resection of ICC (HR =1.454,1.276,1.344,1.588,1.490,1.949,1.574,95％ CI:1.245-1.697,1.056-1.541,1.155-1.565,1.364-1.850,1.235-1.797,1.641-2.314,1.228-2.018,P＜0.05).(7) C-indexof3-,5-year survival probability was 0.74 and 0.71,(95％ CI:0.71-0.77,0.68-0.73).Calibration curve showed that there was a little difference between predicted probability and actual observation value,with a good curve-fitting.There were 456 patients in high score group,456 in median score group and 458 in low score group based on survival risk of nomogram scores.Levels of CA19-9 and CEA,tumor diameter,number of patients with 1 tumor,2 tumors and tumors more than 3 or vessel system invasion,vascular invasion,lymph node metastasis and local invasion were 256.7 U/mL (range,0.7-1 000.0 U/mL),4.6 μg/L (range,0.5-849.6 ug/L),8.0 cm (range,3.0-18.0 cm),194,332,456,113,207,98 in the high score group and 40.7 U/mL(range,1.1-1 000.0 U/mL),2.5 μg/L(range,0.7-45.7 μg/L),6.0 cm(range,2.0-14.8 cm),160,124,2,67,50,19 in the median score groups and 19.5 U/mL(range,0.6-239.1 U/mL),2.1 μg/L(range,0.4-7.5 μg/L),4.6 cm (range,1.2-7.4 cm),102,0,0,0,0,50 in the low score group,respectively,showing statistically significant differences in the above indexes among the 3 groups (Z =128.924,74.923,131.178,x2=146.354,50.298,135.928,60.936,P ＜ 0.05).The 5-year recurrence rate and 5-year overall survival rate in the high,median and low score groups were 93.5％,78.2％,57.4％;9.4％,28.3％,55.6％,respectively,with statistically significant differences among the 3 groups (x2=124.478,161.025,P ＜ 0.05).Conclusions Liver resection produces possible long-term survival in selected patients with ICC.Tumor diameter ＞ 5 cm,multiple tumors,vascular invasion,lymph node metastasis and local invasion are independent risk factors affecting tumor early recurrence after liver resection of ICC.CA19-9 ＞ 39 U/mL,CEA ＞ l0 μg/mL,tumor diameter ＞ 5 cm,multiple tumors,vascular invasion,lymph node metastasis and local invasion are independent risk factors affecting patients' prognosis after liver resection of ICC.The nomogram serves as an accurate stage tool to predict long-term outcomes.

Sorafenib is a multikinase inhibitor approved for the treatment of advanced hepatocellular carcinoma (HCC). However, therapeutic response to sorafenib was not equal among HCC patients. Here we present a novel system to provide quantitative information concerning sorafenib-related targets by simultaneous detection of phosphorylated ERK (pERK) and pAkt expressions in circulating tumor cells (CTCs) isolated from HCC patients. Our results showed that 90.0% of patients had a molecular classification of tissues concordant with that of CTCs. CTC counts showed a shaper decline in patients with pERK+/pAkt- CTCs after two weeks of sorafenib treatment (P < 0.01). Disease control rates were significantly different between patients with pERK+/pAkt- CTCs (11/15; 73.3%) and those without (13/44; 29.5%) (P < 0.05). Univariate and multivariate analysis indicated pERK+/pAkt- CTCs as an independent predictive factor of progression-free survival (PFS) (hazard ratio = 9.389; P < 0.01). PFS correlated with the proportion of pERK+/pAkt- CTCs (r = 0.968, P < 0.01), and was higher in patients with ≥ 40% pERK+/pAkt- CTCs compared to those with < 40% (8.4 vs. 1.3 mo; P < 0.05). In a validation set of twenty HCC patients, CTCs from patients with ≥ 40% pERK+/pAkt- CTCs had significantly higher inhibition rates of spheroid formation compared to those with < 40% (61.2 vs. 19.8%; P < 0.01). Our findings demonstrated that CTCs can be used in place of tumor tissue for characterization of pERK/pAkt expression. pERK+/pAkt- CTCs are most sensitive to sorafenib and an independent predictive factor of PFS in HCC patients treated with sorafenib.