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Background Nonalcoholic fatty liver disease and its advanced stage, nonalcoholic steatohepatitis (NASH), are the major cause of hepatocellular carcinoma (HCC) and other end-stage liver disease. However, the potential mechanism and therapeutic strategies have not been clarified. This study aimed to identify potential roles of miRNA/mRNA axis in the pathogenesis and drug combinations in the treatment of NASH. Methods Microarray GSE59045 and GSE48452 were downloaded from the Gene Expression Omnibus and analyzed using R. Then we obtained differentially expressed genes (DE-genes). DAVID database was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis. Protein-protein interaction (PPI) networks were used for the identification of hub genes. We found upstream regulators of hub genes using miRTarBase. The expression and correlation of key miRNA and its targets were detected by qPCR. Drug Pair Seeker was employed to predict drug combinations against NASH. The expression of miRNA and hub genes in HCC was identified in the Cancer Genome Atlas database and Human Protein Atlas database. Results Ninety-four DE-genes were accessed. GO and KEGG analysis showed that these predicted genes were linked to lipid metabolism. Eleven genes were identified as hub genes in PPI networks, and they were highly expressed in cells with vigorous lipid metabolism. hsa-miR-335-5p was the upstream regulator of 9 genes in the 11 hub genes, and it was identified as a key miRNA. The hub genes were highly expressed in NASH models, while hsa-miR-335-5p was lowly expressed. The correlation of miRNA-mRNA was established by qPCR. Functional verification indicated that hsa-miR-335-5p had inhibitory effect on the development of NASH. Finally, drug combinations were predicted and the expression of miRNA and hub genes in HCC was identified. Conclusions In the study, potential miRNA-mRNA pathways related to NASH were identified. Targeting these pathways may be novel strategies against NASH.

Portal vein involvement is considered one of the most fearful complications of hepatocellular carcinoma (HCC). Portal vein tumor thrombosis (PVTT) is associated with aggressive tumor biology (high grade), high tumor burden (number and size of lesions), high levels of serum markers (AFP), poor liver function (deranged LFT), and poor performance status of patients. The Barcelona Clinic Liver Cancer staging system places HCC patients with PVTT in advanced stage (BCLC Stage-C). This group contains a fairly heterogeneous patient population, previously considered candidates for palliative systemic therapy with sorafenib. However, this provided modest overall survival (OS) benefit. The results of a recent Phase III (IMbrave150) trial favor the combination of atezolizumab and bevacizumab over sorafenib as a standard of care in advanced unresectable HCC. While only lenvatinib proved to be non-inferior against sorafenib in a phase III (REFLECT trial), regorafenib (RESORCE trial), ramucirumab (REACH-2), and cabozantinib (CELESTIAL) have been approved second-line therapy in phase III clinical trials. Recently, the data on the prospect of other modalities in the management of HCC with PVTT is mounting with favorable results. Targeting multiple pathways in the HCC cascade using a combination of drugs and other modalities such as RT, TACE, TARE, and HAIC appear effective for systemic and loco-regional control. The quest for the ideal combination therapy and the sequence set is still widely unanswered and prospective trials are lacking. With the armament of available therapeutic options and the advances and refinements in the delivery system, down-staging patients to make them eligible for curative resection has been reported. In a rapidly evolving treatment landscape, performing surgery when appropriate, in the form of LR and even LT to achieve cure does not seem farfetched. Likewise, adjuvant therapy and prompt management of the recurrences holds the key to prolong OS and DFS. This review discusses the management options of HCC patients with PVTT.

Background: Hepatocellular carcinoma (HCC) is the dominant type of primary liver cancer, which leads to the fourth-ranked common cause of cancer-related death worldwide. The highly heterogeneous tumor microenvironment (TME) contributes to poor prognosis, particularly tumor recurrence of HCC. There is still a great challenge to illuminate the complicated modulation of TME during HCC progression. Methods: In the current study, RNA expression data and clinical-pathological data of HCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The immune and stromal scores of the samples were calculated using ESTIMATE algorithm and DEGs were obtained by comparison of low- and high-score groups. Intersection of hubgenes in protein-protein interaction (PPI) network and factors correlated with survival from univariate COX regression analysis were applied to screen out the key DEGs. The proportion of tumor-infiltrating immune cells (TICs) and stromal components were evaluated using CIBERSORT algorithm. Further Gene Set Enrichment Analysis (GSEA) and correlation analysis were carried out to elucidate the relation between ITK and TIC composition. Findings: The Immune score and Estimate score were proved to be significantly correlated with long-term recurrence-free survival (RFS) of HCC patients. 197 DEGs were carried out based on the Immune and Stromal scores. Two factors including interleukin-2 inducible T-cell kinase (ITK) and HLA Class II histocompatibility antigen DRB5 (HLA-DRB5) were identified as key DEGs. Further analysis proved that ITK was the most powerful DEG to evaluate the clinical-pathological characteristics and outcomes of HCC patients.The results of GSEA revealed that genes in the high ITK subgroup were enriched in inflammatory-immunological terms. CIBERSORT analysis for the proportion of TICs indicated that activated CD4+ memory T cells, CD8+ T cells, and M1 Macrophages were positively correlated with ITK expression, while plasma cells, resting natural killer (NK) cells, activated dendritic cells, activated NK cells, naive CD4+ T cells and resting mast cells were negatively correlated with ITK expression. Interpretation: Our work identified ITK as a novel indicator for tumor recurrence and microenvironment remodeling in HCC, and provide a potential therapeutic target for HCC treatment. Funding Statement: National ST National Natural Science Funds for Distinguished Young Scholar of China (No. 81625003); Key Program, National Natural Science Foundation of China (No. 81930016); Key Research & Development Plan of Zhejiang Province (No. 2019C03050); Youth Program of National Natural Science Foundation of China (No.81702858); Medical and health science and technology program of Zhejiang Province (No.2018256286) Declaration of Interests: None.

BACKGROUND AND OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is highly prevalent in patients who undergo liver transplantation (LT). Whereas there is huge data on NAFLD, little is known about NAFLD in LT. In this review, we aim to explore extrahepatic organs and their potential mechanisms in the development of NAFLD in LT patients and discuss current limitations in preclinical and clinical scenarios with suggestions for future study. METHODS: The following keywords, such as NAFLD, NASH, liver transplant, therapy, pathogenesis and biomarkers, were set for literature retrieval. The articles which were published articles in English till 25th June 2020 in PubMed database were included, and there is no limit for the study design type. KEY CONTENT AND FINDINGS: Following LT, there are significant shifts in the microbiota and farnesoid X receptor may be a potential therapeutic target for NAFLD in LT settings. The roles of probiotics and diet on NALFD remain inconclusive in LT background. Nevertheless, the adipokines and cytokines disorder and local insulin resistance of adipose tissue may contribute to NAFLD process. Bariatric surgeries are promising in controlling de novo and recurrent NAFLD with significant reduction in abdominal adipose tissue, despite the optimal timing is inconclusive in LT cases. Furthermore, circumstantial evidence indicates that miRNA-33a may function as a mediator bridging sarcopenia and NAFLD of post-LT. β-Hydroxy-β-Methyl-Butyrate treatment could improve muscle status in graft recipients and shows protective potential for NAFLD in LT settings. CONCLUSIONS: Gut, adipose tissue and muscle are intricately intertwined in promoting NAFLD in LT cases. Further animal studies are needed to deepen our understanding of mechanisms in multi-organ crosstalk. High quality clinical trials are warrant for making guidelines and developing management strategies on NAFLD after LT.

Background: Early allograft dysfunction (EAD) is associated with decreased graft and patient survival rates. This study aimed to identify the severity of EAD and develop a predictive model for EAD after donation after circulatory death (DCD) liver transplantation (LT). Furthermore, the influence of operative time on EAD incidence was also evaluated. Methods: In this retrospective, multicentre cohort study, nomograms were established based on a single-centre training cohort (n=321) and validated in a 3-center validation cohort (n=501). Results: The incidence rate of EAD was 46.4% (149/321) in the training cohort and 40.5% (203/501) in the validation cohort. Of the 149 EAD patients in the training cohort, 77 patients with either elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) were classified as having EAD type A, and the rest of the EAD patients were classified as having EAD type B. Recipients with EAD type B had lower graft and patient survival rates than recipients with EAD type A (P=0.043 and 0.044, respectively). We further developed a nomogram to predict EAD (graft weight, cold ischemia time, donor age, model for end-stage liver disease (MELD) score) and another nomogram to predict EAD type B (graft weight, cold ischemia time, MELD score). The nomograms for the prediction of EAD and EAD type B had good discrimination [concordance index (C-index) =0.712 (0.666–0.758), 0.707 (0.641–0.773)] and calibration [Hosmer-Lemeshow (HL) P=0.384, P=0.425] in the validation cohort. An increased operative time (>6 h) was associated with increased EAD and EAD type B incidence in the high-risk group (P=0.005, P=0.020, respectively). Conclusions: EAD type B was associated with decreased graft and patient survival rates. The novel nomograms effectively predicted the incidence of EAD and EAD type B in DCD LT patients.

Objective Patient-derived xenograft (PDX) models provide a promising preclinical platform for hepatocellular carcinoma (HCC). However, the molecular features associated with successful engraftment of PDX models have not been revealed. Methods HCC tumor samples from 76 patients were implanted in immunodeficient mice. The molecular expression was evaluated by immunohistochemistry. Patient and tumor characteristics as well as tumor molecular expressions were compared for PDX engraftment using the Chi-square test. The independent prediction parameters were identified by logistic regression analyses. Results The engraftment rate for PDX models from patients with HCC was 39.47% (30/76). Tumors from younger patients and patients with elevated preoperative alpha-fetoprotein level had higher engraftment rates. Tumors with poor differentiation and vascular invasion were related to engraftment success. The positive expression of CK19, CD133, glypican-3 (GPC3), and Ki67 in tumor samples was associated with engraftment success. Logistic regression analyses indicated that GPC3 and Ki67 were two of the strongest predictors of PDX engraftment. Tumors with GPC3/Ki67 phenotypes showed heterogeneous engraftment rates, with 71.9% in GPC3+/Ki67+ tumors, 30.8% in GPC3-/Ki67+ tumors, 15.0% in GPC3+/Ki67- tumors, and 0 in GPC3-/Ki67- tumors. Conclusions Successful engraftment of HCC PDXs was significantly related to molecular features. Tumors with the GPC3+/Ki67+ phenotype were the most likely to successfully establish HCC PDXs.

The downstaging of hepatocellular carcinoma (HCC) has been confirmed to benefit liver transplantation (LT) patients whose tumors are beyond the transplantation criteria. Milan criteria (MC), a tumor size and number-based assessment, is currently used as the endpoint in these patients. However, many studies believe that tumor biological behavior should be added to the evaluation criteria for downstaging efficacy. Hence, this study aimed to explore the feasibility of Hangzhou criteria (HC), which introduced tumor grading and alpha-fetoprotein in addition to tumor size and number, as an endpoint of downstaging.We performed a multicenter and retrospective study of 206 patients accepted locoregional therapy (LRT) as downstaging/bridge treatment prior to LT in three centers of China.Recipients were divided into four groups: failed downstaging to the HC (group A, n = 46), successful downstaging to the HC (group B, n = 30), remained within the HC all the time (group C, n = 113), and tumor progressed (group D, n = 17). The 3-year HCC recurrence probabilities of groups B and C were not significantly different (10.3% vs. 11.6%, P = 0.87). The HCC recurrent rate was significantly higher in group A (52.3%) compared with that in group B/C (P 0.05). Seven patients (7/76, 9.2%) whose tumor exceeded the the HC were successfully downstaged to the MC, and 39.5% (30/76) to the the HC. In group B, 23 patients remained beyond the MC and their survivals were as well as those of patients within the MC.Compared to the MC, HC downstaging criteria can give more HCC patients access to LT and furthermore, the outcome of these patients is the same as those matching MC downstaging criteria. Hangzhou downstaging criteria therefore is applicable in clinical practice.

Aim Glutamine synthetase (GS) levels increase gradually with the development of hepatocellular carcinogenesis. In this study, we aimed to investigate the clinical significance of GS and the underlying mechanism of GS promoting hepatocellular carcinoma (HCC) invasion. Methods Serum concentration of GS and α-fetoprotein (AFP) in HCC patients, liver cirrhosis patients, and healthy individuals were detected. The GS-mRNA level and its prognostic value were explored in an independent HCC cohort from The Cancer Genome Atlas database. GS expression in HCC tissue and matched para-tumor tissue was determined. The effect of GS on HCC invasion was assessed in vitro and in vivo. Results The serum GS and AFP level in HCC patients was higher than that in healthy controls and liver cirrhosis patients. The area under the receiver operating characteristic curve for HCC diagnosis was 0.848 and 0.861 for GS and AFP, respectively. The area under the receiver operating characteristic curve of GS for diagnosis of AFP-negative HCC was 0.913. Combining GS with AFP achieved a diagnostic sensitivity and specificity of 82.5% and 93%, respectively. The GS level was higher in tumor tissues than that in para-tumor tissues. High GS expression was associated with poor prognosis of moderately differentiated HCC patients. In vitro, GS exerted an influence on HCC cell migration by mediating epithelial-mesenchymal transition. The lung and liver metastatic model of HCC further confirmed that GS expression affected the invasion of HCC cells in vivo. Conclusions GS is a useful biomarker for HCC diagnosis, especially for AFP-negative patients. In addition, GS affects HCC metastasis through mediating epithelial-mesenchymal transition.

Background Ubiquitin-specific protease 22 (USP22) is described as a key subunit of the Spt-Ada-Gcn5 acetyl transferase complex, which plays an important role in the prognosis and resistance to chemotherapy drugs in hepatocellular carcinoma (HCC). Silent information regulator 1 (SIRT1) is a member of the sirtuin family that is deubiquitinated by USP22. However, it is still unknown whether USP22 and SIRT1 co-expression is associated with disease progression and 5-Fluorouracil (5-FU) resistance in HCC. Methods 141 patients who received hepatectomy at our hospital from January 2010 to December 2014 were enrolled in this study. The expression of USP22 and SIRT1 was detected by immunohistochemical staining. Clinicopathological features, including age, gender, tumor number, tumor size, tumor differentiation, tumor stage, alpha-fetoprotein and microscopic vascular invasion, were assessed. Further experiments confirmed the role of SIRT1 in 5-FU drug resistance in vivo. Results Immunohistochemical staining showed that the high expression of USP22 and SIRT1 was frequently observed in HCC tissues relative to normal liver tissues. Overexpression of USP22 is associated with microscopic vascular invasion (MVI). Further analysis showed that the co-expression of USP22 and SIRT1 was more effective in predicting the prognosis of HCC. The SIRT1 inhibitor EX-527 dramatically inhibited the expression of Cyclin B1 and resistance-associated protein 3 (MRP3) to reduce 5-FU drug resistance in vivo. Conclusion These findings suggest that the co-expression of USP22 and SIRT1 is significantly associated with unfavorable HCC progression. The inhibition of SIRT1 in vivo could be valuable in improving 5-FU drug sensitivity and inhibiting tumor cell proliferation and inducing apoptosis.

The function of human augmin complex unit 3(Haus3), a component of the HAU augmin-like complex, in various cancers is not clear. This study aims to elucidate the clinical significance and the role of Haus3 in tumor progression of hepatocellular carcinoma (HCC). We analyzed the expression of Haus3 in 50 HCC patients from The Cancer Genome Atlas and 137 HCC patients in our hospital. Compared with adjacent normal tissue, Haus3 expression assessed by immunohistochemical staining was dramatically increased in tumor tissues. A high level of Haus3 expression was significantly correlated with large tumor size (p=0.025) and tumor multiplicity (p=0.004). Univariate and multivariate survival analysis showed thatexpression of Haus3 was an independent prognostic factor for overall survival ofHCCpatients. Western blot analysis showed that Haus3 regulated the phosphorylation of PLK1-T210 and activity of the Cdk1/cyclin B1 complex, indicating that Haus3 disrupted G2/M phase arrest. In immunofluorescence studies, expression of Haus3 correlated with the level ofα-tubulin and γ-tubulin. In summary, Haus3 plays a vital role in regulatingtheactivityof PLK2-T210 and Cdk1/cyclin B1 complex in G2/M phasetransition and the expression of tubulins to ensure normal mitotic progression. Our data suggest that Haus3 might be a promising prognostic biomarker and molecular target of HCC.

Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. Trial register: Trial registered at http://www.chictr.org.cn: ChiCTR2100042869.

Background: Early allograft dysfunction (EAD) after liver transplantation (LT) accompanies poor prognosis of both recipients and grafts. This study aims to explore the relationship between pretransplant intrahepatic protein profiles and the onset of EAD, and the value of combined EAD and protein profiles for predicting recipient and graft survival prognosis. Methods: Liver biopsy specimens of 105 pretransplant grafts used for LT were collected and used for immunohistochemistry analysis of 5 proteins. And matched clinical data of donor, recipient, transplantation and prognosis was analyzed. Findings: The incidence of EAD was 41·9% (44/105) in this cohort. Macrovesicular steatosis (P=0·016), donor body mass index (P=0·013), recipients’ pretransplant serum creatinine (P=0·036), and the intrahepatic expression of heme oxygenase 1 (HO1) (P=0·015) and tumor necrosis factor α (TNF-α) (P=0·039) were independent predictors of the onset of EAD. Inferior graft and recipient prognosis were observed in patients experienced EAD (P=0·028 and 0·031, respectively) or received grafts with higher expression of Sirtuin 1 (P=0·005 and 0·013, respectively). The graft and recipient survival were worst in patients with both EAD development and high expression of Sirtuin 1 (P=0·001 and 0·004, respectively). Interpretation: Pretransplant intrahepatic expression of HO1 and TNF-α are associated with the incidence of EAD. The combination of EAD and EAD-unrelated protein profiles showed superiority in distinguishing recipients with potential worse prognosis. Funding: National Natural Science Foundation of China, Key Research & Development Plan of Zhejiang Province, and Projects of Medical and Health Technology Program in Zhejiang Province. Declaration of Interest: None to declare. Ethical Approval: The procurement of donor grafts was performed in accordance with the national guidelines for DCD in China. The procedure has been described in our previous studies (5). Each organ donation or transplant was approved by the Institutional Review Board, Zhejiang University school of medicine, strictly under the guidelines of the Ethics Committee of the hospital, the current regulation of the Chinese Government, and the Declaration of Helsinki. Informed consent in writing was obtained from each patient. And no organs from executed prisoners were used.

BACKGROUND: Precise recipient selection optimizes the prognosis of liver transplantation (LT) for hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is the most commonly used biomarker for diagnosis and prognosis of HCC in the clinical context. As a crucial molecule in methionine cycle, homocysteine (Hcy) level has been proved to be related to HCC progression and metastasis. OBJECTIVE: We aimed to explore the prognostic capacity of pre-transplant serum Hcy level in LT for HCC. METHODS: This study retrospectively enrolled 161 HCC patients who had underwent LT from donation after cardiac death (DCD) in the First Affiliated Hospital of Zhejiang University from 2015.01.01 to 2018.09.01. Pre-transplant serum Hcy level was incorporated into statistical analysis together with other clinical parameters and pathological features. RESULTS: From an overall perspective, significant difference was observed in Hcy level between recurrence (n= 61) and non-recurrence group (n= 100) though subsequent analysis showed unsatisfactory predicting performance. In the whole cohort, multivariate analysis showed that lnAFP (p= 0.010) and Milan criteria (MC, p< 0.001) were independent risk factors of HCC recurrence after LT. MA score based on MC and lnAFP performed well in predicting post-LT tumor recurrence with the AUROC at 0.836 (p< 0.001) and 3-year recurrence-free survival rate at 96.8% (p< 0.001) in the low risk group (n= 69). According to the clinical practice, serum concentration lower than 20 μg/L is considered as normal range of AFP. Elevated pre-transplant serum AFP (> 20 μg/L) predicts high HCC recurrence after LT. We further divided the 161 recipients into AFP- group (n= 77, AFP ⩽ 20 μg/L) and AFP+ group (n= 84, AFP > 20 μg/L). MA score was still well presented in the AFP+ group and the AUROC for tumor recurrence was 0.823 (p< 0.001), whereas the predicting accuracy was reduced in AFP- group (AUROC: 0.754, P< 0.001). After subsequent analysis, we found that elevated pre-transplant Hcy level (> 12.75 μmol/L) predicted increased tumor recurrence risk in AFP- group. The 3-year recurrence-free survival rates were 92.0% and 53.7% (p< 0.001) in low Hcy subgroup (n= 40) and high Hcy subgroup (n= 37) respectively. Multivariate analysis showed that Hcy (p= 0.040) and Milan criteria (p= 0.003) were independent risk factors for post-transplant tumor recurrence in AFP- group. Further combination of Hcy level and Milan criteria identified a subgroup of AFP- recipients with acceptable outcomes even though beyond Milan criteria (3-year recurrence-free survival rate: 77.7%, p< 0.001). CONCLUSION: As a classic predictor in HCC prognosis, AFP performed well in our study cohort when combined with Milan criteria. Homocysteine was an effective prognostic biomarker in LT for AFP- hepatocellular carcinoma. In recipients exceeding Milan criteria, acceptable post-transplant outcome could be seen in those with low Hcy and AFP level.

Hepatocellular carcinoma (HCC) is one of the most severe diseases worldwide. For the different stages of HCC, there are different clinical treatment strategies, such as surgical therapy for the early stage, and transarterial chemoembolization (TACE) and selective internal radiation therapy (SIRT) for intermediate-stage disease. Systemic treatment, which uses mainly targeted drugs, is the standard therapy against advanced HCC. Sorafenib is an important first-line therapy for advanced HCC. As a classically effective drug, sorafenib can increase overall survival markedly. However, it still has room for improvement because of the heterogeneity of HCC and acquired resistance. Scientists have reported the acquired sorafenib resistance is associated with the anomalous expression of certain genes, most of which are also related with HCC onset and development. Combining sorafenib with inhibitors targeting these genes may be an effective treatment. Combined treatment may not only overcome drug resistance, but also inhibit the expression of carcinoma-related genes. This review focuses on the current status of sorafenib in advanced HCC, summarizes the inhibitors that can combine with sorafenib in the treatment against HCC, and provides the rationale for clinical trials of sorafenib in combination with other inhibitors in HCC. The era of sorafenib in the treatment of HCC is far from over, as long as we find better methods of medication.

CR6-interacting factor 1 (CRIF1) regulates cell cycle progression and the DNA damage response. Here, we show that CRIF1 expression is decreased in hepatocellular carcinoma (HCC) tissues and positively correlates with patients' survival. In vitro, down-regulation of CRIF1 promotes HCC cell proliferation and invasiveness, while over-expression has the opposite effect. in vivo, CRIF1 knockdown enhances growth of HCC xenografts. Analysis of mRNA microarrays showed that CRIF1 knockdown activates genes involved in TGF-β RI/Smad2/3 signaling, leading to epithelial-mesenchymal transition (EMT) and increased matrix metalloproteinase-3 (MMP3) expression. However, cell invasion and EMT are abrogated in HCC cells treated with SB525334, a specific TGF-β RI inhibitor, which indicates the inhibitory effect of CRIF1 on HCC tumor growth is mediated by TGF-β signaling. These results demonstrate that CRIF1 benefits patient survival by inhibiting HCC cell invasiveness through suppression of TGF-β-mediated EMT and MMP3 expression. This suggests CRIF1 may serve as a novel target for inhibiting HCC metastasis.

// Qi Ling 1, 2, * , Xiao Xu 1, 2, * , Panpan Ye 3, * , Haiyang Xie 1, 2 , Feng Gao 1, 2 , Qichao Hu 2 , Zhikun Liu 1, 2 , Xuyong Wei 1, 2 , Christian Roder 4 , Anna Trauzold 4 , Holger Kalthoff 4 , Shusen Zheng 1, 2 1 Department of Surgery, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2 Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, China 3 The Ophthalmology Center, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 4 Institute for Experimental Cancer Research, Comprehensive Cancer Center North, CAU, Kiel, Germany * These authors contributed equally to this work Correspondence to: Shusen Zheng, email: zyzss@zju.edu.cn Holger Kalthoff, email: hkalthoff@email.uni-kiel.de Keywords: anatomy, microRNA, pancreatic cancer, recurrence Received: January 22, 2016 Accepted: January 10, 2017 Published: January 20, 2017 ABSTRACT Different clinical presentations and prognoses have been implied between pancreatic head and body/tail cancers. We aimed to identify the prognostic relevance of primary tumor location in patients undergoing resection for pancreatic ductal adenocarcinoma (PDAC). Thirty-two pairs of patients with strictly matched early stage (II) pancreatic head and body/tail cancers were enrolled. The molecular feature of the two subtypes of PDAC was assessed on the level of miRNA expression. Out of the 64 patients, 34 (53.1%) had tumor recurrence after radical resection during the follow-up period (2.3 ± 0.8 years). Both overall and tumor-free survival were significantly higher in the patients with pancreatic body/tail cancer compared with those with pancreatic head cancer. Patient age and tumor location were the independent prognostic factors for tumor recurrence. A remarkably lower expression of miR-501-3p and higher expression of miR-375 were found and were further verified in pancreatic body/tail cancer tissues compared with pancreatic head cancer tissues. The low expression of miR-501-3p was significantly associated with a low risk of tumor recurrence. Both, subcutaneous and orthotopic PDAC mouse models presented highly invasive tumor phenotypes upon up-regulated miR-501-3p expression. An in vitro study showed that miR-501-3p promoted the invasiveness of PDAC cells possibly via suppressing E-cadherin. In summary, at resectable early stage, pancreatic body/tail cancer presents a less malignant phenotype associated with deregulation of miR-501-3p compared with pancreatic head cancer.

Liver cirrhosis results from many forms of chronic damage, characterized by accumulation of extracellular matrix. The present study aimed to explore a potential non-invasive biomarker and its mechanism in the progression of liver cirrhosis.Gene Expression Omnibus (GEO) dataset (GSE15654, n = 216) was analyzed to screen genes associated with progression of liver cirrhosis. A total of 181 plasma samples, including healthy control (HC, n = 20), chronic hepatitis B (CHB, n = 77) and HBV-related liver cirrhosis (LC, n = 84), were enrolled for validation. In vitro and in vivo experiments were employed for the mechanistic investigation.GEO dataset analysis showed that relatively low mRNA-expression of CC motif chemokine ligand 16 (CCL16) was associated with elevated Child-Pugh score (P = 0.034) and worse prognosis (P = 0.025). Plasma CCL16 level decreased in a stepwise pattern, with a median concentration of 10.29, 6.57 and 4.47 ng/mL in the HC, CHB and LC groups, respectively (P0.001). Low plasma CCL16 was significantly related to hepatic dysfunction both in the CHB and LC groups (P0.05). Combination of CCL16 and ALT showed improved distinguishing capability for LC compared to either alone. In vitro, CCL16 expression was downregulated by lipopolysaccharide and hypoxia. Overexpression of CCL16 from human normal liver cell line (LO2) reduced the extracellular matrix associated proteins (Col1 and Col4) in human hepatic stellate cell line (LX-2). In vivo, the pathological feature of cirrhosis was alleviated by the hepatocyte-specific expression of CCL16.CCL16 could be a feasible plasma marker to predict the occurrence and progression of liver cirrhosis. CCL16 might impact liver cirrhosis through inactivating hepatic stellate cells.

Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is a significant clinical problem associated with poor surgical outcomes. This study aims to summarize the current evidence on risk prediction models of HCC recurrence after LT. PubMed and EMBASE were searched to May 25, 2019, for relevant articles. Studies originally designed to develop or validate a risk prediction model for HCC recurrence after LT were included. Two independent authors summarized the study characteristics and evaluated the risk of bias and applicability concerns in the included studies. From 26 included studies, 18 original risk prediction models were determined, but only five models were externally validated. The average number of predictors involved in the construction of risk models was three. The most frequently employed predictors were alpha-fetoprotein, tumor size, vascular invasion, tumor number, tumor differentiation, and neutrophil-lymphocyte ratio. Most studies showed good discriminatory performance (AUC >0.75). The overall quality of the included studies was generally low. Most of the original models lacked the highly recommended external and prospective validation in diverse populations. The AFP model was the well-validated preoperative risk model that can stratify patients into high- and low-risk groups.

BACKGROUND Early recurrence after liver transplantation (LT) is still a clinical problem. This multicenter study evaluated the Milan, Hangzhou, and AFP model-based criteria for prediction of early recurrence of HCC in patients with cirrhosis who had undergone LT. MATERIAL AND METHODS From the China Liver Transplant Registry (CLTR) database, we analyzed data of 589 HCC patients who had undergone LT between Jan 2015 and Jan 2019. Imaging data and AFP levels were evaluated immediately before LT. Recurrence and overall survival rates at 2 years were tested using the Kaplan-Meier estimate. The Milan criteria, Hangzhou criteria, and AFP model-based criteria were evaluated. RESULTS We found that 62.0%, 91.2%, and 67.6% of patients were within the Milan criteria, Hangzhou criteria, and AFP model-based criteria, respectively. The 2-year recurrence rate was 8.9%, 15.8%, and 11.8% with corresponding overall survival of 85.3%, 82.7%, and 86.5%, respectively. The 2-year recurrence rate was different in patients fulfilling and exceeding the AFP model-based criteria among patients who met either the Milan criteria (7.9% vs. 18.8%, HR=3.83, p=0.006) or Hangzhou criteria (12.0% vs. 27.6%, HR=2.95, p

Background Early recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is associated with poor surgical outcomes. This study aims to construct a preoperative model to predict individual risk of post-LT HCC recurrence. Methods Data of 748 adult patients who underwent deceased donor LT for HCC between January 2015, and February 2019 were collected retrospectively from the China Liver Transplant Registry database and randomly divided into training (n = 486) and validation(n = 262) cohorts. A multivariate analysis was performed and the five-eight model was developed. Results A total of 748 patients were included in the study; of them, 96% had hepatitis B virus (HBV) and 84% had cirrhosis. Pre-LT serum alpha-fetoprotein (AFP), tumor number and largest tumor diameter were incorporated to construct the 5–8 model which can stratify patients accurately according to their risk of recurrence into three prognostic subgroups; low-(0–5 points), medium-(6–8 points) and high-risk (> 8 points) with 2-year post-LT recurrence rate of (5,20 and 51%,p Conclusions The 5–8 model is a simple validated and accurate tool for preoperative stratification of early recurrence of HCC after LT.

Resistance to sorafenib remains one of the biggest concerns for hepatocellular carcinoma treatment. In article number 2003042, Xiao Xu, Jiajia Xiang, and co‐workers developed a self‐activated sorafenib and USP22 shRNA codelivery nanoplatform (Gal‐SLP) to reverse cancer stemness and sensitive cancer cells to sorafenib. Gal‐SLP releases USP22 shRNA efficiently and enhances the intracellular sorafenib accumulation in cancer cells. Gal‐SLP exhibits potent antitumor efficiency in a sorafenibinsensitive patient‐derived xenograft (PDX) model. [Image: see text]

We aim to evaluate the efficacy and safety of basiliximab in liver transplantation (LT) for patients with hepatitis B virus-related diseases.A total of 268 patients with hepatitis B virus-related diseases undergoing LT were enrolled and divided into two groups according to the usage of basiliximab. Total survival, the survival of high-risk patients defined by the posttransplant model for predicting mortality, acute rejection rate, biochemical parameters and other follow-up data were compared between the two groups.Group Bas was composed of 131 patients who received basiliximab, and Group Triple enrolled the other 137 patients who did not. Between the two groups, there was no significant difference in the cumulative survival of patients without hepatocellular carcinoma (HCC) or in the cumulative survival of patients with HCC. For patients with benign end-stage liver diseases, Group Bas had more patients with a high risk of short- and medium-term mortality than Group Triple (22.81% vs. 8.85%, p = 0.017), but the survival curves of the two groups were not significantly different. The 1-year incidence of acute rejection was lower in Group Bas, although the difference was not significant (8.75% vs. 15.33%, p0.05). In both Group Bas and Group Triple, the level of serum creatinine (Scr) at 1 week posttransplantation was significantly lower than pretransplantation (61.00 vs. 88.50 μmol/l, p0.001; 61.50 vs. 74.00 μmol/l, p0.001; respectively). There was a significant difference in the pretransplantation Scr between the two groups (88.50 vs. 74.00 μmol/l, p = 0.005), but the values of Scr decreased to the same level 1 week (61.00 vs. 61.50 μmol/l, p0.05) and 4 weeks (61.00 vs. 59.00 μmol/l, p0.05) after transplantation. Significantly fewer recipients in Group Bas experienced hepatitis B relapse than in Group Triple (2/131 vs. 13/137, p = 0.006).A basiliximab-induced immunosuppressive protocol is a safe regimen that achieves similar survival without increasing the acute rejection rate for LT recipients with hepatitis B virus-related diseases. For patients with benign end-stage liver diseases, this regimen reduces medium-term mortality in high-risk patients. This regimen remarkably improves renal function in the first month after LT and is correlated with a decreased hepatitis B recurrence rate in adult patients after LT.

Objective Liver transplantation is an optimal radical therapy for selected patients with hepatocellular carcinoma. The stringent organ allocation system driven by the Milan criteria has been challenged by alternative sets of expanded criteria. Careful analysis is needed to prove that the Milan criteria can be expanded safely and effectively. Design This study collectively reviewed 6012 patients of hepatocellular carcinoma from the China Liver Transplant Registry. Expanded criteria were evaluated to characterise an optimised expansion with acceptable outcomes beyond the Milan criteria. Results Compared with the Milan criteria, Valencia, University of California, San Francisco, University Clinic of Navarra and Hangzhou criteria provided an expansion of 12.4%, 16.3%, 19.6%, and 51.5%, respectively. The post-transplant survivals of patients fulfilling the expanded criteria were comparable to that of the Milan criteria. The analysis of net reclassification improvement and area under the receiver operating characteristic curves showed an excellent efficiency in recurrence prediction for the expanded criteria compared with the Milan criteria. In patients exceeding Milan but fulfilling the Hangzhou criteria (N=1352), α-fetoprotein (AFP) >100 ng/mL and tumour burden>8 cm were the only two independent prognostic factors (p8 cm but AFP≤100 ng/mL) and type B (tumour burden >8 cm but AFP between 100 and 400 ng/mL). Type A showed significantly higher 5-year tumour-free survival rates compared with type B (p

Background: Hepatic hemangiomas are the most common benign liver tumors, and the management of giant hepatic hemangioma (GHH) is still in controversial. The aim of this meta-analysis was to compare the postoperative outcomes of enucleation versus anatomic resection for GHH. Methods: PubMed, Embase, Web of Science, and the Cochrane Library were searched from January 1988 to December 2015 to identify studies comparing the outcomes of enucleation versus anatomic resection for GHH. Finally, we performed this meta-analysis using the Review Manager 5.3 software, and the results were presented as risk ratio (RR) or mean difference (MD) with corresponding 95% confidence interval (CI). The major limitation is that all data were derived from nonrandomized studies, and we cannot exclude potential selection bias. Results: Nine studies involving 1,185 patients were included. The results showed that there was a lower incidence of complications (RR = 0.66, 95% CI 0.52 to 0.84, I2 = 0%, p = 0.0007); no incidents of death occurred among the 9 included trials. Blood loss (MD = -419.07 mL, 95% CI -575.04 to -263.09, I2 = 83%, p < 0.00001), duration of surgery (MD = -23.5 min, 95% CI -45.28 to -1.74, I2 = 0%, p = 0.03), and length of hospital stay (MD = -1.59 days, 95% CI -3.06 to -0.13, I2 = 0%, p = 0.03) were much lower in the enucleation group. Conclusions: GHH can be removed safely by either enucleation or anatomic resection. Enucleation can preserve more hepatic parenchyma and reduce postoperative complications, which is why it should be the preferred surgical procedure for suitable lesions.

Background Four tumor markers for hepatocellular carcinoma (HCC), alpha-fetoprotein (AFP), glypican-3 (GPC3), vascular endothelial growth factor (VEGF) and des-gamma-carboxy prothrombin (DCP), are closely associated with tumor invasion and patient's survival. This study estimated the predictability of preoperative tumor marker levels along with pathological parameters on HCC recurrence after hepatectomy. Methods A total of 140 patients with HCC who underwent hepatectomy between January 2012 and August 2012 were enrolled. The demographics, clinical and follow-up data were collected and analyzed. The patients were divided into two groups: patients with macroscopic vascular invasion (MaVI+) and those without MaVl (MaVI-). The predictive value of tumor markers and clinical parameters were evaluated by univariate and multivariate analysis. Results In all patients, tumor size (>8 cm) and MaVI were closely related to HCC recurrence after hepatectomy. For MaVI+ patients, VEGF (>900 pg/mL) was a significant predictor for recurrence (RR=2.421; 95% CI: 1.272–4.606; P=0.007). The 1- and 2-year tumor-free survival rates for MaVI+ patients with VEGF ≤900 pg/mL versus for those with VEGF >900 pg/mL were 51.5% and 17.6% versus 19.0% and 4.8% (P>0.001). For MaVI-patients, DCP >445 mAu/mL and tumor size >8 cm were two independent risk factors for tumor recurrence (RR=2.307, 95% CI: 1.132–4.703, P=0.021; RR=3.150, 95% CI: 1.392–7.127, P=0.006; respectively). The 1- and 2-year tumor-free survival rates for the patients with DCP ≤445 mAu/mL and those with DCP >445 mAu/mL were 90.4% and 70.7% versus 73.2% and 50.5% respectively (P=0.048). The 1- and 2-year tumor-free survival rates for the patients with tumor size ≤8 cm and >8 cm were 83.2% and 62.1% versus 50.0% and 30.0%, respectively (P=0.003). Conclusions The MaVI+ patients with VEGF ≤900 pg/mL had a relatively high tumor-free survival than those with VEGF >900 pg/mL, In the MaVI-patients, DCP >445 mAu/mL and tumor size >8 cm were predictive factors for postoperative recurrence.

Background and Aim: Portal vein tumor thrombus (PVTT) is one of the factors that can affect prognosis and survival of hepatocellular carcinoma (HCC). In the present study, we aimed to find out some biomarkers associated with vascular invasion features of HCC with the method of comparative proteomic analysis. Methods: The proteins were extracted from a pair of HCC tissues with PVTT and without PVTT, and then separated by two-dimensional polyacrylamide gel electrophoresis. Differentially expressed protein spots were identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Further analysis of two proteins were completed using real-time fluorescence quantitative polymerase chain reaction and western-blot in 40 HCC tissues with PVTT (n = 20) and without PVTT (n = 20). Results: Among 465 protein spots displayed on the gels, 33 unique proteins (> twofold change, P

Preexisting diabetes mellitus (DM) has been reported to have an adverse consequence on patient prognosis after liver transplantation in the West. So far, there are few reports on the effect of preexisting DM on outcome of liver transplant recipients with HBV infection. We aimed to examine the impact of preexisting DM on post-transplant outcome in Chinese patients with HBV-related liver disease. The post-transplant morbidities and patient survival were compared between 48 diabetes patients (DM group) and 96 non-diabetes patients (control group) matched for age, gender, primary disease and model for end-stage liver diseases score. The DM group was further divided into hyperglycemia patients (fasting blood glucose >8.0 mmol/L, n = 22) and non-hyperglycemia patients (fasting blood glucose ≤8.0 mmol/L, n = 26). Patient characteristics were comparable between both groups, except a higher incidence of hepatic encephalopathy in the DM group than that in the control group (22.9% vs. 10.4%, P = 0.045). The incidences of post-transplant complications and patient survival did not differ significantly between the DM and control groups, or between non-hyperglycemia patients and their matched case controls. Hyperglycemia patients showed a higher incidence of post-transplant sepsis (18.2% vs. 2.3%, P = 0.039) and biliary complications (31.8% vs. 6.8%, P = 0.012) than their matched case controls. Preexisting DM is not a contraindication for liver transplantation in patients with HBV-related liver disease. A tight control of blood glucose to a level of ≤8.0 mmol/L was necessary to reduce the risk of complications after liver transplantation.

Background Needle core biopsy (NCB) is one of the most widely used and accepted methods for the diagnosis of focal hepatic lesions. Although many studies have assessed the diagnostic accuracy of NCB in predicting the tumor grade, it is still under debate. Objective To identify the influence of number of biopsies on NCB diagnostic accuracy. Methods 153 patients with HCC were selected from patients who received preoperative NCB under the guidance of ultrasonography in our hospital. The diagnostic reference standard was the surgical pathologic diagnosis. Results Using a 3-tier grading scheme (well, moderate and poor), the accuracy of NCB has no significant differences among different number of passes in HCC ≤5cm. For HCC >5≤8cm, the increasing number of passes could increase the diagnostic accuracy (63.3%, 81.8%, and 84.8% for passes one, two, and three, respectively). While in HCC>8cm, the diagnostic accuracy of passes one, two, and three were 62.1%, 69%, and 75.8%, respectively. Conclusions The accuracy of NCB in assessing tumor grading associated with tumor size and number of passes. Meanwhile, a minimum of two passes should be performed to get better accuracy in patients with HCC >5cm.

Background. Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide. It is characterized by its high invasive and metastatic potential. Leprecan-like 1 (LEPREL1) has been demonstrated to be downregulated in the HCC tissues in previous proteomics studies. The present study is aimed at a new understanding of LEPREL1 function in HCC.Methods. Quantitative RT-PCR, immunohistochemical analysis, and western blot analysis were used to evaluate the expression of LEPREL1 between the paired HCC tumor and nontumorous tissues. The biology function of LEPREL1 was investigated by Cell Counting Kit-8 (CCK8) assay and colony formation assay in HepG2 and Bel-7402 cells.Results. The levels of LEPREL1 mRNA and protein were significantly lower in the HCC tissues as compared to those of the nontumorous tissues. Reduced LEPREL1 expression was not associated with conventional clinical parameters of HCC. Overexpression of LEPREL1 in HepG2 and Bel-7402 cells inhibited cell proliferation (P<0.01) and colony formation (P<0.05). LEPREL1 suppressed tumor cell proliferation through regulation of the cell cycle by downregulation of cyclins.Conclusions. Clinical parameters analysis suggested that LEPREL1 was an independent factor in the development of HCC. The biology function experiments showed that LEPREL1 might serve as a potential tumor suppressor gene by inhibiting the HCC cell proliferation.

Backgrounds and aims Accurate assessment of graft bile duct is important to plan surgical procedure. Magnetic resonance cholangiopancreatography (MRCP) has become an important diagnostic procedure in evaluation of pancreaticobiliary ductal abnormalities and has been reported as highly accurate. We aim to estimate the efficacy of preoperative MRCP on depicting biliary anatomy in living donor liver transplantation (LDLT), and to determine whether inaccurate preoperative imaging assessment would increase the biliary complications after LDLT. Methods The data of 118 cases LDLT were recorded. Information from preoperative MRCP was assessed using intraoperative cholangiography (IOC) as the gold standard. The possible risk factors of recipient biliary complications were analyzed. Results Of 118 donors, 84 had normal anatomy (type A) and 34 had anatomic variants (19 cases of type B, 9 cases of type C, 1 case of type E, 2 cases of type F and 3 cases of type I) confirmed by IOC. MRCP correctly predicted all 84 normal cases and 17 of 34 variant cases, and showed an accuracy of 85.6% (101/118). The incidence of biliary complications was comparable between cases with accurate and inaccurate classification of biliary tree from MRCP, and between cases with normal and variant anatomy of bile duct. While cases with graft duct opening ≤5mm showed a significant higher incidence of total biliary complications (21.1% vs. 6.6%, P =0.028) and biliary stricture (10.5% vs. 1.6%, P =0.041) compared with cases with large duct opening >5mm. Conclusion MRCP could correctly predict normal but not variant biliary anatomy. Inaccurate assessment of biliary anatomy from MRCP not increases the rate of biliary complications, while small-sized graft duct may cause an increase in biliary complications particularly biliary stricture after LDLT.