Hypoxia-driven tumor stromal remodeling and immunosuppressive microenvironment in scirrhous HCC.

BACKGROUND AND AIMS: Scirrhous HCC (SHCC) is one of the unique subtypes of HCC, characterized by abundant fibrous stroma in the tumor microenvironment. However, the molecular traits of SHCC remain unclear, which is essential to develop specialized therapeutic approaches for SHCC. APPROACH AND RESULTS: We presented an integrative analysis containing single-cell RNA-sequencing, whole-exome sequencing, and bulk RNA-sequencing in SHCC and usual HCC samples from 134 patients to delineate genomic features, transcriptomic profiles, and stromal immune microenvironment of SHCC. Multiplexed immunofluorescence staining, flow cytometry, and functional experiments were performed for validation. Here, we identified SHCC presented with less genomic heterogeneity while possessing a unique transcriptomic profile different from usual HCC. Insulin-like growth factor 2 was significantly upregulated in SHCC tumor cells compared to usual HCC, and could serve as a potential diagnostic biomarker for SHCC. Significant tumor stromal remodeling and hypoxia were observed in SHCC with enrichment of matrix cancer-associated fibroblasts and upregulation of hypoxic pathways. Insulin-like growth factor 2 was identified as a key mediator in shaping the hypoxic stromal microenvironment of SHCC. Under this microenvironment, SHCC exhibited an immunosuppressive niche correlated to enhanced VEGFA signaling activity, where CD4 + T cells and CD8 + T cells were dysfunctional. Furthermore, we found that another hypoxic-related molecule SPP1 from SHCC tumor cells suppressed the function of dendritic cells via the SPP1-CD44 axis, which also probably hindered the activation of T cells. CONCLUSION: We uncovered the genomic characteristics of SHCC, and revealed a hypoxia-driven tumor stroma remodeling and immunosuppressive microenvironment in SHCC.

FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. METHODS: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. RESULTS: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. CONCLUSIONS: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs. IMPACT AND IMPLICATIONS: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs.

Anti-PD-1 antibody in combination with radiotherapy as first-line therapy for unresectable intrahepatic cholangiocarcinoma.

BACKGROUND: Unresectable intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis despite treatment with standard combination chemotherapy. We aimed to evaluate the efficacy and safety of radiotherapy in combination with an anti-PD-1 antibody in unresectable iCCA without distant metastases. METHODS: In this phase II study, patients with histopathologically confirmed unresectable primary or postoperative recurrent iCCA without distant metastases were enrolled. Patients received external radiotherapy with a dose of ≥45 Gy (2-2.5 Gy per fraction), followed by anti-PD-1 immunotherapy (camrelizumab 200 mg once, every 3 weeks) initiated within 7 days after completion of radiotherapy as first-line therapy. The primary endpoint was 1-year progression-free survival (PFS) rate. The secondary end points included safety, objective response rate (ORR), disease control rate (DCR), and overall survival (OS). RESULTS: From December 2019 to March 2021, 36 patients completed radiotherapy and at least one cycle of immunotherapy and were included in efficacy and safety analyses. The median follow-up was 19.0 months (IQR 12.0-24.0), and the one-year PFS rate was 44.4% (95% CI, 30.8-64.0). The median PFS was 12.0 months (95% CI, 7.5-not estimable); the median OS was 22.0 months (95% CI, 15.0-not estimable). The ORR was 61.1% and the DCR was 86.1%. Seventeen of 36 (47.2%) patients experienced treatment-related adverse effects (AEs) of any grade. The most common AE was reactive cutaneous capillary endothelial proliferation (25.0%). Five (13.9%) patients experienced grade ≥3 treatment-related AEs, including decreased lymphocyte (5.6%), bullous dermatitis (2.8%), decreased platelet count (2.8%), and deep-vein thrombosis (2.8%). CONCLUSIONS: External radiotherapy plus camrelizumab, as first-line therapy, met its primary endpoint and showed antitumor activity and low toxicity levels in patients with unresectable iCCA without distant metastases, warranting further investigation. TRIAL REGISTRATION: NCT03898895. Registered 2 April 2019.

Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. DESIGN: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. RESULTS: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. CONCLUSIONS: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

Distinct single-cell immune ecosystems distinguish true and de novo HBV-related hepatocellular carcinoma recurrences.

OBJECTIVE: Revealing the single-cell immune ecosystems in true versus de novo hepatocellular carcinoma (HCC) recurrences could help the optimal development of immunotherapies. DESIGN: We performed 5'and VDJ single-cell RNA-sequencing on 34 samples from 20 recurrent HCC patients. Bulk RNA-sequencing, flow cytometry, multiplexed immunofluorescence, and in vitro functional analyses were performed on samples from two validation cohorts. RESULTS: Analyses of mutational profiles and evolutionary trajectories in paired primary and recurrent HCC samples using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The tumour immune microenvironment (TIME) of truly recurrent HCCs was characterised by an increased abundance in KLRB1+CD8+ T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8+ T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed elevated GDF15 expression on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited antitumour immunity in truly recurrent lesions. In contrast, myeloid cells' cross talk with T cells-mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCCs. Consistent with these findings, a phase 2 trial of neoadjuvant anti-PD-1 immunotherapy showed more responses in de novo recurrent HCC patients. CONCLUSION: True and de novo HCC recurrences occur early, have distinct TIME and may require different immunotherapy strategies. Our study provides a source for genomic diagnosis and immune profiling for guiding immunotherapy based on the type of HCC recurrence and the specific TIME.

Integrative metabolomic characterisation identifies altered portal vein serum metabolome contributing to human hepatocellular carcinoma.

OBJECTIVE: Altered metabolites are important for the tumourigenicity of hepatocellular carcinoma (HCC). We performed integrative metabolomics analysis of the metabolites changes in portal venous blood and in comparison with the metabolites changes in liver tissues and stool samples of HCC patients and healthy liver donors. DESIGN: Serum (portal and central vein), liver tissue (HCC tumour and adjacent non-tumour, normal liver) and stool samples were collected from 102 subjects (52 HCC patients and 50 healthy controls) in the discovery cohort; and 100 subjects (50 HCC patients and 50 healthy controls) in an independent validation cohort. Untargeted metabolomic profiling was performed using high-performance liquid chromatography-mass spectrometry. The function of candidate metabolites was validated in hepatocyte cell lines. RESULTS: Detailed metabolomic evaluation showed distinct clusters of metabolites in serum, liver tissue and stool samples from patients with HCC and control individuals (p<0.001). HCC patients had significantly higher levels of portal vein serum and HCC tissue metabolites of DL-3-phenyllactic acid, L-tryptophan, glycocholic acid and 1-methylnicotinamide than healthy controls, which were associated with impaired liver function and poor survival. On the other hand, HCC patients had lower levels of linoleic acid and phenol in portal vein and stool samples than healthy controls. Linoleic acid and phenol significantly inhibited HCC proliferation, inferring their anti-HCC function as protective metabolites. CONCLUSIONS: The integrative metabolome analysis of serum, tissue and stool metabolites revealed unreported metabolic alterations in HCC patients. In portal vein, we identified elevated and depleted metabolites signifying that they might play a role in HCC development.

Role of Preoperational Imaging Traits for Guiding Treatment in Single ≤ 5 cm Hepatocellular Carcinoma.

BACKGROUND: Imaging traits including nonsmooth tumor margins, internal arteries, peritumoral enhancement, and absence of hypodense halos can reflect tumor aggressiveness preoperatively and may affect treatment selection. This study aimed to explore the role of these four imaging traits in treatment selection between surgical resection (SR) and radiofrequency ablation (RFA) for patients with single ≤ 5 cm hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Three hundred eight-one patients with single ≤ 5 cm HCC who underwent SR (n = 202) or RFA (n = 179) in the First Affiliated Hospital of Sun Yat-sen University from April 2010 to December 2019 were retrospectively enrolled. The efficacy of SR and RFA in patients with the imaging traits that significantly influenced recurrence-free survival (RFS) or overall survival (OS) was compared and analyzed. RESULTS: Multivariable Cox regression analysis identified that having internal arteries (P = 0.001) was an independent influencing factor for RFS, while internal arteries (P = 0.005) and peritumoral enhancement (P = 0.001) were independent influencing factors for OS. In patients with internal arteries, subgroup analysis based on tumor size demonstrated that both RFS and OS of SR were superior to those of RFA in patients with 3-5 cm HCC (RFS, P = 0.023; OS, P = 0.015). In patients with peritumoral enhancement, both RFS and OS of SR were superior to those of RFA (RFS, P = 0.019; OS, P = 0.042). CONCLUSION: SR may be associated with improved survival compared with RFA in patients with single 3-5 cm HCC having internal arteries and patients with single ≤ 5 cm HCC having peritumoral enhancement.

Preoperative Survival Prediction in Intrahepatic Cholangiocarcinoma Using an Ultrasound-Based Radiographic-Radiomics Signature.

OBJECTIVES: To construct a preoperative model for survival prediction in intrahepatic cholangiocarcinoma (ICC) patients using ultrasound (US) based radiographic-radiomics signatures. METHODS: Between April 2010 and September 2015, 170 patients with ICC who underwent curative resection were retrospectively recruited. Overall survival (OS)-related radiographic signatures and radiomics signatures based on preoperative US were built and assessed through a time-dependent receiver operating characteristic curve analysis. A nomogram was developed based on the selected predictors from the radiographic-radiomics signatures and clinical and laboratory results of the training cohort (n = 127), validated in an independent testing cohort (n = 43) by the concordance index (C-index), and compared with the Tumor Node Metastasis (TNM) cancer staging system as well as the radiographic and radiomics nomograms. RESULTS: The median areas under the curve of the radiomics signature and radiographic signature were higher than that of the TNM staging system in the testing cohort, although the values were not significantly different (0.76-0.82 versus 0.62, P = .485 and .264). The preoperative nomogram with CA 19-9, sex, ascites, radiomics signature, and radiographic signature had C-indexes of 0.72 and 0.75 in the training and testing cohorts, respectively, and it had significantly higher predictive performance than the 8th TNM staging system in the testing cohort (C-index: 0.75 versus 0.67, P = .004) and a higher C-index than the radiomics nomograms (0.75 versus 0.68, P = .044). CONCLUSIONS: The preoperative nomogram integrated with the radiographic-radiomics signature demonstrated good predictive performance for OS in ICC and was superior to the 8th TNM staging system.

Contrast-Enhanced Ultrasound-Based Nomogram: A Potential Predictor of Individually Postoperative Early Recurrence for Patients With Combined Hepatocellular-Cholangiocarcinoma.

PURPOSES: To evaluate the postsurgical prognostic implication of contrast-enhanced ultrasound (CEUS) for combined hepatocellular-cholangiocarcinoma (CHC). To build a CEUS-based early recurrence prediction classifier for CHC, in comparison with tumor-node-metastasis (TNM) staging. METHODS: The CEUS features and clinicopathological findings of each case were analyzed, and the Liver Imaging Reporting and Data System categories were assigned. The recurrence-free survival associated factors were evaluated by Cox proportional hazard model. Incorporating the independent factors, nomograms were built to estimate the possibilities of 3-month, 6-month, and 1-year recurrence and whose prognostic value was determined by time-dependent receiver operating characteristics, calibration curves, and hazard layering efficiency validation, comparing with TNM staging system. RESULTS: In the multivariable analysis, the levels of carbohydrate antigen 19-9, prothrombin time and total bilirubin, and tumor shape, the Liver Imaging Reporting and Data System category were independent factors for recurrence-free survival. The LR-M category showed longer recurrence-free survival than did the LR-4/5 category. The 3-month, 6-month, and 1-year area under the curves of the CEUS-clinical nomogram, clinical nomogram, and TNM staging system were 0.518, 0.552, and 0.843 versus 0.354, 0.240, and 0.624 (P = .048, .049, and .471) vs. 0.562, 0.545, and 0.843 (P = .630, .564, and .007), respectively. The calibration curves of the CEUS-clinical model at different prediction time pionts were all close to the ideal line. The CEUS-clinical model effectively stratified patients into groups of high and low risk of recurrence in both training and validation set, while the TNM staging system only works on the training set. CONCLUSIONS: Our CEUS-clinical nomogram is a reliable early recurrence prediction tool for hepatocellular-cholangiocarcinoma and helps postoperative risk stratification.

Risk predictive model based on three immune-related gene pairs to assess prognosis and therapeutic sensitivity for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) as a common tumor has a poor prognosis. Recently, a combination of atezolizumab and bevacizumab has been recommended as the preferred regimen for advanced HCC. However, the overall response rate of this therapy is low. There is an urgent need to identify sensitive individuals for this precise therapy among HCC patients. METHODS: The Wilcox test was used to screen the differentially expressed immune-related genes by combining the TCGA cohort and the Immunology Database. Univariate and multivariate Cox regression analysis were used to screen the immune gene pairs concerning prognosis. A predictive model was constructed using LASSO Cox regression analysis, and correlation analysis was conducted between the signature and clinical characteristics. ICGC cohort and GSE14520 were applied for external validations of the predictive risk model. The relationship between immune cell infiltration, TMB, MSI, therapeutic sensitivity of immune checkpoint inhibitors, targeted drugs, and the risk model were assessed by bioinformatics analysis in HCC patients. RESULTS: A risk predictive model consisting of 3 immune-related gene pairs was constructed and the risk score was proved as an independent prognostic factor for HCC patients combining the TCGA cohort. This predictive model exhibited a positive correlation with tumor size (p < 0.01) and tumor stage (TNM) (p < 0.001) in the chi-square test. The predictive power was verified by external validations (ICGC and GSE14520). The risk score clearly correlated with immune cell infiltration, MSI, immune checkpoints, and markers of angiogenesis. CONCLUSIONS: Our research established a risk predictive model based on 3 immune-related gene pairs and explored its relationship with immune characteristics, which might help to assess the prognosis and treatment sensitivity to immune and targeted therapy of HCC patients.

Regional liver function analysis with gadoxetic acid-enhanced MRI and virtual hepatectomy: prediction of postoperative short-term outcomes for HCC.

OBJECTIVES: To explore the role of quantitative regional liver function assessed by preoperative gadoxetic acid-enhanced MRI with computer-aided virtual hepatectomy to predict short-term outcomes after major hepatectomy for HCC. METHODS: We retrospectively reviewed the records of 133 consecutive patients with HCC who underwent preoperative gadoxetic acid-enhanced MRI and indocyanine green (ICG) test. Forty-five patients received open major hepatectomy. Liver function reserve and the future liver remnant were evaluated by computer-aided virtual hepatectomy. Global liver functional parameters included the T1 relaxation time reduction rate (T1ratio) and functional liver volume (FV), whereas regional parameters included the rT1pos, rT1ratio, remnant FV (rFV), and remnant FV ratio (rFVratio) of the remnant liver. The functional parameters of the MRI and ICG were used to predict the short-term outcomes (liver failure and major complications) after major hepatectomy. RESULTS: The T1ratio and FV were correlated with the ICG test (rho = - 0.304 and - 0.449, p < 0.05). FV < 682.8 ml indicated preoperative ICG-R15 ≥ 14% with 0.765 value of the area under the curve (AUC). No patient who underwent major resection with good liver functional reserve (ICG < 14%) and enough future remnant volume (> 30% standard LV) developed liver failure. Low rT1ratio (< 66.5%) and high rT1pos (> 217.5 ms) may predict major complications (AUC = 0.831 and 0.756, respectively; p < 0.05). The rT1ratio was an independent risk factor for postoperative major complications (odds ratio [OR] = 0.845, 95% CI, 0.736-0.966; p < 0.05). CONCLUSION: Preoperative gadoxetic acid-enhanced MRI with computer-aided virtual hepatectomy may facilitate optimal assessment of regional liver functional reserve to predict short-term outcomes after major hepatectomy for HCC. KEY POINTS: • Preoperative gadoxetic acid-enhanced MRI with virtual hepatectomy and volumetric analysis can provide precise liver volume and regional functional assessment. • Quantitative regional liver function assessed by gadoxetic acid-enhanced MRI can predict the short-term outcomes after major hepatectomy in patients with HCC. • The regional liver function assessed by gadoxetic acid-enhanced MRI is an independent risk factor for postoperative major complications.

Kinesin family member 2C aggravates the progression of hepatocellular carcinoma and interacts with competing endogenous RNA.

Kinesin family member 2C (KIF2C), a substantial mitotic regulator, has been verified to exert a malignant function in several cancers. However, its function in hepatocellular carcinoma (HCC) remains unclear. In this study, the expression profile of KIF2C in HCC was characterized through the dataset from the TCGA and clinical tissue microarrays containing 220 pairs of resected HCC tissues and adjacent nontumor tissues in our hospital. The results indicated that KIF2C was substantially higher expression in tumor tissues than adjacent nontumor tissues. High expression of KIF2C significantly correlated with large tumor (>5.0 cm) (P = .001) and implied a dismal postoperative overall survival (OS) (hazard ratio [HR] = 1.729; P = .002) in our cohort of patients. Gain and loss of function assays displayed that KIF2C promoted HCC cell proliferation, accelerated cell cycle progression, and impeded apoptosis. By bioinformatic tools and mechanistic investigation, we found that KIF2C interacted with various cell-cycle-related proteins and was significantly involved in growth-promoting pathways. KIF2C upregulated PCNA and CDC20 expression. Subsequently, we investigated the regulation of KIF2C by competing endogenous RNA and elucidated that has-miR-6715a-3p was directly bond to the 3'-untranslated region of KIF2C through dual luciferase assays, thereby inhibiting KIF2C expression. Furthermore, the long noncoding RNA GS1-358P8.4 was found to be a candidate of KIF2C for has-miR-6715a-3p binding. HCC patients with high lncRNA-GS1-358P8.4 expression had shorter OS and relapse-free survival compared to those with low expression, which was accordance with the KIF2C. Taken together, KIC2C aggravated HCC progression, it could serve as a prognostic indicator and confer a novel target for clinical treatment.

The prognostic value of neuromedin U in patients with hepatocellular carcinoma.

BACKGROUND: Neuromedin U (NMU) is a neuropeptide belonging to the neuromedin family. Recently, significant associations between NMU and several cancers have been reported. However, no studies have examined the association between NMU and hepatocellular carcinoma (HCC). The purpose of this study was to examine the role of NMU in HCC. METHODS: An enzyme-linked immunosorbent assay was used to measure the level of NMU protein in the sera of patients with hepatic hemangioma and HCC. NMU and cytokine mRNA expression was assessed in HCC samples via RT-qPCR. A tissue microarray consisting of 228 HCC peri- and intra-tumor tissues was used to detect NMU expression via immunohistochemical analysis. The association between NMU expression and overall survival (OS) and disease-free survival (DFS) was analyzed by Kaplan-Meier curves, the log-rank test, and Cox proportional hazard model. RESULTS: The level of NMU protein was increased in the sera of HCC patients (p = 0.006). NMU was expressed in intercellular space, rather than in hepatocytes or HCC cells. The prognosis of HCC patients with high NMU expression in peri-tumor tissue was significantly poorer than that of patients with low NMU expression (OS: p = 0.002, DFS: p = 0.033). Peri-tumor NMU expression was also a significant independent prognostic factor for OS (hazard ratio: 1.541, 95% confidence interval: 1.092-2.175, p = 0.014). The level of NMU expression was positively associated with M2 macrophage percentage and the levels of type-2 inflammatory cytokines in HCC tissue. CONCLUSIONS: NMU may serve as a novel prognostic biomarker for HCC patients, although further validation is needed in the future. The activation of M2 macrophages and a type-2 inflammatory response may involve in the role of NMU in patients with HCC.

Comparison between M-score and LR-M in the reporting system of contrast-enhanced ultrasound LI-RADS.

OBJECTIVE: To develop a contrast-enhanced ultrasound (CEUS) M-score and compare it with LR-M in CEUS Liver Imaging Reporting and Data System (LI-RADS). METHODS: We retrospectively enrolled 105 consecutive high-risk patients with hepatocellular carcinoma (HCC) and 105 with intrahepatic cholangiocarcinoma (ICC). The subjects were selected by propensity score matching between November 2003 and December 2017. A CEUS M-score for predicting ICC was constructed based on specific CEUS features by the least absolute shrinkage and selection operator regularised regression. M-score was used to develop a modified CEUS LI-RADS. The diagnostic performance of the modified CEUS LI-RADS using M-score for diagnosing HCC and ICC was compared with American College of Radiology (ACR) CEUS LI-RADS using LR-M. RESULTS: The most useful features for ICC were as follows: poorly circumscribed (69.52%), rim enhancement (63.81%), early washout (92.38%), intratumoural vein (56.19%), obscure boundary of intratumoural non-enhanced area (57.14%), and marked washout (59.05%, all p < 0.001). For predicting ICC, the M-score had a higher specificity (88.57% vs. 63.81%) with lower sensitivity (89.52% vs. 95.24%) compared with LR-M. For diagnosing HCC, the sensitivity of modified LI-RADS (80.95%) was much higher than that of ACR LI-RADS (57.14%), but the specificity was lower (90.48% vs. 96.19%). The area under the curve (AUC) of modified LI-RADS (0.857) was much higher than that of ACR LI-RADS (0.767, p = 0.0001). The modified positive predictive value (PPV) of ACR LI-RADS and modified LI-RADS were 99.42% and 98.99%, respectively. CONCLUSIONS: The modified LI-RADS with M-score had higher sensitivity for diagnosing HCC and higher specificity for diagnosing ICC than ACR LI-RADS. KEY POINTS: • For predicting ICC, the M-score had a higher specificity (88.57% vs. 63.81%) with lower sensitivity (89.52% vs. 95.24%) compared with LR-M. • A CEUS M-score for predicting ICC consisted of more detailed CEUS features (poorly circumscribed, rim enhancement, early washout, intratumoural vein, obscure boundary of intratumoural non-enhanced area, and marked washout) was constructed. • For diagnosing HCC, the sensitivity of modified LI-RADS (80.95%) was much higher than that of ACR LI-RADS (57.14%), but the specificity was lower (90.48% vs. 96.19%). The modified positive predictive value (PPV) of ACR LI-RADS and modified LI-RADS were 99.42% and 98.99%, respectively.

Hornerin promotes tumor progression and is associated with poor prognosis in hepatocellular carcinoma.

BACKGROUND: The function of hornerin (HRNR), a member of the S100 protein family, is poorly clarified in the development of human tumors. The role of HRNR in hepatocellular carcinoma (HCC) progression is investigated in the study. METHODS: The expression levels of HRNR were assessed in tumor samples from a cohort of 271 HCC patients. The effect of HRNR on proliferation, colony formation and invasion of tumor cells was examined. We further determined the role of HRNR in tumor growth in vivo by using xenograft HCC tumor models. The possible mechanism of the HRNR promotion of HCC progression was explored. RESULTS: We found that HRNR was overexpressed in HCC tissues. The high expression of HRNR in HCCs was significantly associated with vascular invasion, poor tumor differentiation, and advanced TNM stage. The disease-free survival (DFS) and overall survival (OS) of HCC patients with high HRNR expression were poorer than those in the low HRNR expression group. HRNR expression was an independent risk factor linked to both poor DFS (HR = 2.209, 95% CI = 1.627-2.998,P <  0.001) and OS (HR = 2.459,95% CI = 1.736-3.484, P <  0.001). In addition, the knockdown of HRNR by shRNAs significantly inhibited the proliferation, colony formation, migration and invasion of HCC tumor cells. HRNR silencing led to the decreased phosphorylation of AKT signaling. Notably, tumor growth was markedly inhibited by HRNR silencing in a xenograft model of HCC. CONCLUSIONS: HRNR promotes tumor progression and is correlated with a poor HCC prognosis. HRNR may contribute to HCC progression via the regulation of the AKT pathway.

Screening for immune-potentiating antigens from hepatocellular carcinoma patients after radiofrequency ablation by serum proteomic analysis.

BACKGROUND: Radiofrequency ablation (RFA) can not only effectively kill hepatocellular carcinoma (HCC) tumour cells but also release tumour antigens that can provoke an immune response. However, there is no consensus regarding which antigens could constitutively be generated after RFA and could potentiate the immune response. The aim of this study was to identify these immune-potentiating antigens. METHODS: We performed two-dimensional electrophoresis (2-DE) and MALDI-TOF-MS/MS analyses on serum obtained before and after RFA from 5 HCC patients. Further validation for selected proteins was performed utilizing ELISA analysis on another 52 HCC patients. Disease-free survival (DFS) analysis according to the differential expression of the interested protein before and after RFA was performed. RESULTS: Twelve decreased and 6 increased proteins after RFA were identified by MS. Three proteins, including clusterin, Ficolin-3, and serum retinol binding protein-4, were further verified by ELISA on the 52 HCC patients. Only Ficolin-3 proved to be significantly changed after RFA. The 52 patients were divided into two groups according to the expression of Ficolin-3 before and after RFA. The 1-, 2- and 3-year DFS rates were 59.1%, 31.8%, and 22.7%, respectively, for patients in the low Ficolin-3 group (22 patients) and 73.3%, 60.0%, and 50.0%, respectively, for patients in the high Ficolin-3 group (30 patients) (P = 0.038). CONCLUSIONS: In conclusion, Ficolin-3 was overexpressed in the serum of most HCC patients after RFA. Ficolin-3 might be a biomarker for RFA treatment efficacy and a potential target for HCC immunotherapy.

Low expression of c-Myc protein predicts poor outcomes in patients with hepatocellular carcinoma after resection.

BACKGROUND: Embryonic Liver Fodrin (ELF) is an adaptor protein of transforming growth factor (TGF-ß) signaling cascade. Disruption of ELF results in mislocalization of Smad3 and Smad4, leading to compromised TGF-ß signaling. c-Myc is an important oncogenic transcription factor, and the disruption of TGF-ß signaling promotes c-Myc-induced hepatocellular carcinoma (HCC) carcinogenesis. However, the prognostic significance of c-Myc in HCC is less understood METHODS: The expression of c-Myc protein and mRNA were measured by immunohistochemistry (IHC) and qRT- PCR, respectively. IHC was performed to detect TGF-ß1 and ELF expression in HCC tissues. Their relationship with clinicopathological factors and overall survival (OS) and disease free survival (DFS) were examined. RESULTS: The expression of c-Myc protein and mRNA in HCC tissues were significantly higher in HCC area than those in normal liver tissues. However, the expression were low compared with those adjacent to HCC area. c-Myc protein was independently predictive of DFS and OS, and it was negatively correlated with tumor size (P = 0.031), tumor number (P = 0.038), and recurrence (P = 0.001). Low c-Myc expression was associated with short-term recurrence and poor prognosis. The predictive value of c-Myc combined with TGF-ß1 or/and ELF was higher than that of any other single marker. Low c-Myc, high TGF-ß1 or/and low ELF expression was associated with the worst DFS and OS. CONCLUSIONS: Low expression of c-Myc protein predicts poor outcomes in patients with HCC with hepatectomy. The combination of the expression of c-Myc, TGF-ß1, and ELF can be used to accurately predict outcomes of patients with HCC.

Non-Invasive Diagnostic Criteria for Hepatocellular Carcinoma in Hepatitis B Virus-Endemic Areas: Is Cirrhosis Indispensable?

AIM: To confirm whether cirrhosis is indispensable for the non-invasive diagnostic criteria for hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-endemic areas. METHODS: Between January 2014 and December 2014, a total of 409 patients with pathologically proven focal liver lesions who underwent contrast-enhanced ultrasound (CEUS) were recruited from our institution. Clinical liver cirrhosis, HBV/HCV infection and HCC-typical vascular pattern of the targeted lesion on CEUS were evaluated. The following 3 criteria were applied to these patients to diagnose HCC: criterion 1, clinical liver cirrhosis and HCC-typical vascular pattern; criterion 2, HBV/HCV infection and HCC-typical vascular pattern; criterion 3, HBV/HCV infection or clinical liver cirrhosis and HCC-typical vascular pattern. Pathological reports were considered the gold standard. RESULTS: A total of 311 patients had confirmed HCC by pathology. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value and area under the ROC curve for criterion 1 were 29.6, 90.8, 44.3, 91.1, 28.9, and 0.60% respectively. For criterion 2, they were 83.3, 74.5, 81.2, 91.2, 58.4, and 0.79%, respectively, and for criterion 3, they were 86.2, 72.5, 82.9, 90.9, 62.3, and 0.79% respectively. CONCLUSIONS: In HBV-endemic areas, when using the HBV/HCV infection instead of cirrhosis as the precondition of the non-invasive diagnostic criteria for HCC, we should be aware of the potential false positive. Cirrhosis still plays an important role in the non-invasive diagnostic criteria for HCC because of the high specificity.

Theranostical nanosystem-mediated identification of an oncogene and highly effective therapy in hepatocellular carcinoma.

UNLABELLED: Because the primary surgical treatment options for hepatocellular carcinoma (HCC)-including hepatic resection and liver transplantation-often fail due to recurrence and metastasis, identifying early prognostic biomarkers and therapeutic targets for HCC is of great importance. This study shows that transducin ß-like protein 1-related protein (TBLR1) is a key HCC oncogene that plays important roles in HCC proliferation, antiapoptosis, and angiogenesis by regulating the Wnt/ß-catenin pathway. The folate-targeted theranostic small interfering RNA (siRNA) nanomedicine Fa-PEG-g-PEI-SPION/psiRNA-TBLR1 effectively silences the TBLR1 gene in different human HCC cell lines in vitro and in human HCC samples in vivo, resulting in the simultaneous suppression of HCC cell proliferation, antiapoptosis, and angiogenesis. Because of its multi-anticancer functions against HCC, intravenous injection of the folate-targeted siRNA nanomedicine into nude mice bearing intrahepatic or subcutaneous xenografts of human HCC has a significant therapeutic effect. Tumor growth in those animals was almost completely inhibited by treatment with Fa-PEG-g-PEI-SPION/psiRNA-TBLR1. Moreover, the SPION-encapsulated polyplexes possess high magnetic resonance imaging (MRI) detection sensitivity, which makes tumor-targeted siRNA delivery easily trackable using the clinical MRI technique. CONCLUSION: The theranostic siRNA nanomedicine examined here possesses great theranostic potential for combined gene therapy and MRI diagnosis of HCC.

Thrombocytopenia and the outcomes of hepatectomy for hepatocellular carcinoma: a meta-analysis.

BACKGROUND: Recently, increasing studies have revealed the association of inflammatory parameters, such as preoperative platelet count, and the prognosis of hepatocellular carcinoma (HCC). However, the link between the platelet count and the prognosis of patients with HCC after hepatic resection is still controversial. METHODS: We searched PubMed, Web of Science, EMBASE, and CBM for relevant trials and analyzed outcomes with random-effects model. The hazard ratio (HR) and its 95% confidence interval (CI) were calculated. RESULTS: In total, 31 studies, including a total of 10,730 patients, met our criteria. The results showed that thrombocytopenia in HCC patients was associated with poor overall survival (HR = 1.47, 95% CI: 1.21-1.78), disease-free survival (HR = 1.36, 95% CI: 1.08-1.72), and a high risk of cancer recurrence (HR = 1.41, 95% CI: 1.22-1.62), but a low risk of extrahepatic metastasis (HR = 0.55, 95% CI: 0.47-0.63). CONCLUSIONS: The meta-analysis revealed that preoperative platelet count could act as a significant biomarker in the prognosis of HCC, especially a platelet count of <100 × 103/mm3. Additional high-quality trials are needed, considering the low-quality studies analyzed.