Sortilin-Driven Cancer Secretome Enhances Tumorigenic Properties of Hepatocellular Carcinoma via de Novo Lipogenesis.

A growing body of evidence suggests de novo lipogenesis as a key metabolic pathway adopted by cancers to fuel tumorigenic processes. While increased de novo lipogenesis has also been reported in hepatocellular carcinoma (HCC), understanding on molecular mechanisms driving de novo lipogenesis remains limited. In the present study, the functional role of sortilin, a member of the vacuolar protein sorting 10 protein receptor family, in HCC was investigated. Sortilin was overexpressed in HCC and was associated with poorer survival outcome. In functional studies, sortilin-overexpressing cells conferred tumorigenic phenotypes, namely, self-renewal and metastatic potential, of HCC cells via the cancer secretome. Proteomic profiling highlighted fatty acid metabolism as a potential molecular pathway associated with sortilin-driven cancer secretome. This finding was validated by the increased lipid content and expression of fatty acid synthase (FASN) in HCC cells treated with conditioned medium collected from sortilin-overexpressing cells. The enhanced tumorigenic properties endowed by sortilin-driven cancer secretome were partly abrogated by co-administration of FASN inhibitor C75. Further mechanistic dissection suggested protein stabilization by post-translational modification with O-GlcNAcylation as a major mechanism leading to augmented FASN expression. In conclusion, the present study uncovered the role of sortilin in hepatocarcinogenesis via modulation of the cancer secretome and deregulated lipid metabolism.

Stanniocalcin 1 is a serum biomarker and potential therapeutic target for HBV-associated liver fibrosis.

Stanniocalcin 1 (STC1), a secreted protein, is upregulated in human cancers including hepatocellular carcinoma (HCC). While most HCCs develop from chronic liver disease, which involves progressive parenchymal injury and fibrosis, the role of STC1 in this preneoplastic stage remains poorly understood. In this study we investigated the clinical relevance and functional significance of secreted STC1 in liver fibrosis. To this end, the STC1 level was determined in the serum samples of chronic hepatitis B patients and correlated with the degree of liver fibrosis. Diagnostic performance of STC1 was analysed by area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, positive predictive value, and negative predictive value. The results were compared with other well-characterised serum biomarkers for liver fibrosis: Aspartate transaminase to Platelet Ratio Index (APRI) and Fibrosis-4 (FIB-4). The functional role of STC1 was interrogated by in vitro experiments using cell line models. Expression of fibrogenic markers was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results showed that the serum STC1 level in chronic hepatitis B patients was positively correlated with the degree of liver fibrosis and showed a stepwise increase in accordance with the severity of fibrosis. The AUROCs for detecting significant fibrosis (>9.0 kPa) and cirrhosis (>12.0 kPa) was 0.911 and 0.880, respectively. STC1 demonstrated a superior specificity and positive predictive value when compared to APRI and FIB-4. Consistent with this, STC1 was elevated in the liver tissues and sera of CCl4 -treated mice showing marked liver fibrosis. In vitro, STC1 was secreted by the human hepatic stellate cell line LX2. Human recombinant STC1 (rhSTC1) induced expression of fibrogenic markers in LX2 cells. The profibrogenic phenotype conferred by rhSTC1 or TGF-ß1 in LX2 cells could be attenuated using anti-STC1 antibody. Taken together, STC1 is a specific serum biomarker for HBV-associated liver fibrosis. STC1 functionally promotes liver fibrogenesis and is a potential actionable target. © 2022 The Pathological Society of Great Britain and Ireland.

Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.

OBJECTIVE: We examined the serological, virological (in serum and liver) and histological profiles in chronic hepatitis B virus (HBV) patients during and after completion of multiple dose (MD) ARC-520. DESIGN: The present phase 1b study was a multidose, open-label extension cohort of patients that had received single dose ARC-520 in our previous study. Eight patients received 4-9 4 weekly doses of MD ARC-520 and entecavir. Liver biopsies were performed in six patients. Intrahepatic and serum HBV DNA, HBV RNA and viral antigens were measured. RESULTS: All patients had 28.9-30.4 months of follow-up after the last MD. All three hepatitis B e antigen (HBeAg)-positive patients had profound reductions in hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen and HBV RNA with two undergoing HBeAg seroconversion. One further achieved HBsAg seroconversion (anti-HBs level of 25.1 IU/L) and the remaining two had HBsAg reductions of -1.7 and -3.5 log IU/mL >30 months after MD. Among the five HBeAg-negative patients, four had modest HBsAg reduction >29 months after completion of MD and one achieved HBsAg seroconversion (anti-HBs level of 152.5 IU/L) and was negative for liver HBsAg staining. Entecavir was successfully stopped in this patient 12 months after HBsAg seroconversion. Temporally related alanine aminotransferase elevations preceded by HBsAg reductions were observed in three patients suggesting immune activation. HBcAg staining was negative in all six biopsied patients. Two patients with <10% HBsAg positive staining of hepatocytes had correspondingly low serum HBsAg levels of 1.5 and 11.5 IU/mL. CONCLUSIONS: MD ARC-520 therapy achieved sustained and profound reductions of viral antigens and HBV RNA. HBsAg seroclearance was achievable. TRIAL REGISTRATION NUMBER: NCT02065336.

Anti-tumour effects of PIM kinase inhibition on progression and chemoresistance of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a biologically aggressive cancer. Targeted therapy is in need to tackle challenges in the treatment perspective. A growing body of evidence suggests a promising role of pharmacological inhibition of PIM (proviral integration site for Moloney murine leukaemia virus) kinase in some human haematological and solid cancers. Yet to date, the potential application of PIM inhibitors in HCC is still largely unexplored. In the present study we investigated the pre-clinical efficacy of PIM inhibition as a therapeutic approach in HCC. Effects of PIM inhibitors on cell proliferation, migration, invasion, chemosensitivity, and self-renewal were examined in vitro. The effects of PIM inhibitors on tumour growth and chemoresistance in vivo were studied using xenograft mouse models. Potential downstream molecular mechanisms were elucidated by RNA sequencing (RNA-seq) of tumour tissues harvested from animal models. Our findings demonstrate that PIM inhibitors SGI-1776 and PIM447 reduced HCC proliferation, metastatic potential, and self-renewal in vitro. Results from in vivo experiments supported the role of PIM inhibition in suppressing of tumour growth and increasing chemosensitivity of HCC toward cisplatin and doxorubicin, the two commonly used chemotherapeutic agents in trans-arterial chemoembolisation (TACE) for HCC. RNA-seq analysis revealed downregulation of the MAPK/ERK pathway upon PIM inhibition in HCC cells. In addition, LOXL2 and ICAM1 were identified as potential downstream effectors. Taken together, PIM inhibitors demonstrated remarkable anti-tumourigenic effects in HCC in vitro and in vivo. PIM kinase inhibition is a potential approach to be exploited in formulating adjuvant therapy for HCC patients of different disease stages. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Among Patients with Undetectable Hepatitis B Surface Antigen and Hepatocellular Carcinoma, a High Proportion Has Integration of HBV DNA into Hepatocyte DNA and No Cirrhosis.

BACKGROUND & AIMS: In some individuals with undetectable serum levels of hepatitis B surface antigen (HBsAg), hepatitis B virus (HBV) DNA can still be detected in serum or hepatocytes and HBV replicates at low levels-this is called occult HBV infection (OBI). OBI has been associated with increased risk of hepatocellular carcinoma (HCC). We investigated the incidence of OBI in patients with HCC and other liver diseases. We also investigated whether, in patients with OBI and HCC, HBV DNA has integrated into the DNA of hepatocytes. METHODS: We collected clinical information and liver tissues from 110 HBsAg-negative patients (90 with HCC and 20 without HCC; median ages at surgical resection and biopsy collection, 64.1 and 48.6 years, respectively) who underwent liver resection or liver biopsy from November 2002 through July 2017 in Hong Kong. HBV DNA and covalently closed circular DNA (cccDNA) were analyzed and quantified by PCR in liver tissues. Integration of HBV DNA into the DNA of liver cells was detected by Alu-PCR. RESULTS: Of the 90 HBsAg-negative patients with HCC, 18 had alcoholic liver disease (20%), 14 had non-alcoholic fatty liver disease or steatohepatitis (16%), 2 had primary biliary cholangitis, 2 had recurrent pyogenic cholangitis, 1 had autoimmune hepatitis, and 53 had none of these (59%). Among the 20 patients without HCC, 7 had non-alcoholic fatty liver disease or steatohepatitis, 7 had primary biliary cholangitis, and 6 had autoimmune hepatitis. OBI was detected in 62/90 patients with HCC (69%) and 3/20 patients without HCC (15%) (P < .0001). cccDNA was detectable in liver cells of 29 patients with HCC and OBI (47%) and HBV DNA had integrated into DNA of liver cells of 43 patients with HCC and OBI (69%); cccDNA and integrated HBV DNA were not detected in the 3 patients who had OBI without HCC. There were 29 patients with integration of HBV DNA among 33 patients with undetectable cccDNA in liver tissues (88%) and 14 patients with integration of HBV DNA among the 29 patients with cccDNA in liver tissues (48%) (P = .001). HBV DNA was found to integrate near genes associated with hepatocarcinogenesis, such as those encoding telomerase reverse transcriptase, lysine methyltransferase 2B, and cyclin A2. Among the 43 patients with integration of HBV DNA, 39 (91%) did not have cirrhosis. CONCLUSIONS: In an analysis of clinical data and liver tissues from 90 HBsAg-negative patients with HCC, we found that almost 70% had OBI, of whom 70% had integration of HBV DNA into liver cell DNA; 90% of these patients did not have cirrhosis. HBV DNA integrated near hepatic oncogenes; these integrations might promote development of liver cancer.

Deregulated GATA6 modulates stem cell-like properties and metabolic phenotype in hepatocellular carcinoma.

Accumulating evidence illustrates the significance of cell plasticity in the molecular biology of liver cancer. Reprogramming of mature parenchymal cells to a less differentiated state by key molecular targets contributes to the pathogenesis of hepatocellular carcinoma (HCC). Hereby, we investigated the role of GATA6, a transcription factor implicated in hepatocyte lineage specification, in HCC. Our results demonstrated a lower expression of GATA6 in HCC tissues compared to the corresponding nontumoral liver tissues. Moreover, GATA6 underexpression, as observed in about 50% cases in our clinical cohort, was associated with a poorer degree of tumor cell differentiation and worse disease-free survival outcome. In vitro, silencing of GATA6 in HCC cells augmented cell migration and invasion abilities of HCC cells by activating epithelial-mesenchymal transition. Self-renewal was also enhanced in vitro. Consistently, in vivo tumorigenicity and self-renewal was promoted upon GATA6 knockdown. Notably, suppression of GATA6 converts HCC cells to a metabolic phenotype recapitulating stem-cell state. Expression of glycolytic markers was elevated in GATA6-knockdown clones accompanied by increased glucose uptake; while overexpression of GATA6 resulted in opposite effects. Further to this, we identified that GATA6 bound to the promoter region of PKM gene and regulated PKM2 transcription. Taken together, downregulation of GATA6 directs HCC cells to glycolytic metabolism and fosters tumorigenicity, self-renewal and metastasis. GATA6 is a transcriptional regulator and a genetic switch that converts the phenotypic reprogramming of HCC cells. It is a potential prognostic biomarker and therapeutic target for liver cancer.

Expression of hepatic progenitor cell markers in acute cellular rejection of liver allografts-An immunohistochemical study.

BACKGROUND: Hepatic progenitor cells (HPC) are induced following liver injury to facilitate regeneration. Acute cellular rejection (ACR) is a common complication after liver transplantation as a result of immune-mediated liver injury. In this study, we characterized HPC phenotype in liver allograft biopsy with ACR. We also explored the correlation between expression HPC immunophenotype and clinicopathological parameters. METHODS: Forty-four liver allograft biopsies performed between 2008 and 2016 in a single center with histologically proven ACR were examined for immunohistochemical expression of HPC markers CK19 and Sox9. The number of positive-staining cells was assessed and correlated with clinicopathological features by statistical analysis. RESULTS: HPC phenotype expression as denoted by CK19 and Sox9 staining was detected in the liver tissue with ACR. The numbers of CK19+ and Sox9+ cells were positively correlated. A larger number of CK19+ cells were associated with higher serum aspartate aminotransferase (AST) level at biopsy. By histological rejection score, a larger number of Sox9+ cells were associated with a higher score of bile duct damage. CONCLUSION: Expression HPC markers were correlated with clinical and histological parameters in ACR. Expression of each individual marker may be more tightly associated with a particular component of the ACR process.

Deregulation of Frizzled Receptors in Hepatocellular Carcinoma.

G protein-coupled receptors (GPCRs) have a substantial role in tumorigenesis and are described as a "cancer driver". Aberrant expression or activation of GPCRs leads to the deregulation of downstream signaling pathways, thereby promoting cancer progression. In hepatocellular carcinoma (HCC), the Wnt signaling pathway is frequently activated and it is associated with an aggressive HCC phenotype. Frizzled (FZD) receptors, a family member of GPCRs, are known to mediate Wnt signaling. Accumulating findings have revealed the deregulation of FZD receptors in HCC and their functional roles have been implicated in HCC progression. Given the important role of FZD receptors in HCC, we summarize here the expression pattern of FZD receptors in HCC and their corresponding functional roles during HCC progression. We also further review and highlight the potential targeting of FZD receptors as an alternative therapeutic strategy in HCC.

SENP1 promotes hypoxia-induced cancer stemness by HIF-1α deSUMOylation and SENP1/HIF-1α positive feedback loop.

OBJECTIVE: We investigated the effect and mechanism of hypoxic microenvironment and hypoxia-inducible factors (HIFs) on hepatocellular carcinoma (HCC) cancer stemness. DESIGN: HCC cancer stemness was analysed by self-renewal ability, chemoresistance, expression of stemness-related genes and cancer stem cell (CSC) marker-positive cell population. Specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) mRNA level was examined with quantitative PCR in human paired HCCs. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation assay to detect the binding of HIFs with hypoxia response element sequence. In vivo characterisation was performed in immunocompromised mice and stem cell frequency was analysed. RESULTS: We showed that hypoxia enhanced the stemness of HCC cells and hepatocarcinogenesis through enhancing HIF-1α deSUMOylation by SENP1 and increasing stabilisation and transcriptional activity of HIF-1α. Furthermore, we demonstrated that SENP1 is a direct target of HIF-1/2α and a previously unrecognised positive feedback loop exists between SENP1 and HIF-1α. CONCLUSIONS: Taken together, our findings suggest the significance of this positive feedback loop between HIF-1α and SENP1 in contributing to the increased cancer stemness in HCC and hepatocarcinogenesis under hypoxia. Drugs that specifically target SENP1 may offer a potential novel therapeutic approach for HCC.

Stearoyl-CoA desaturase regulates sorafenib resistance via modulation of ER stress-induced differentiation.

BACKGROUND & AIMS: We investigated the functional role and clinical significance of stearoyl-CoA desaturase-1 (SCD1) mediated endoplasmic reticulum (ER) stress in regulating liver tumor-initiating cells (T-ICs) and sorafenib resistance, with the aim of developing a novel therapeutic strategy against hepatocellular carcinomas (HCCs). METHODS: We evaluated the clinic-pathological relevance of SCD1 and its correlation with sorafenib resistance in large cohorts of HCC clinical samples by qPCR and immunohistochemical analyses. Lentiviral-based overexpression and knockdown approaches were performed to characterize the functional roles of SCD1 in regulating liver T-ICs and sorafenib resistance. Molecular pathways mediating the phenotypic alterations were identified through RNA sequencing analysis and functional rescue experiments. The combinatorial effect of SCD1 inhibition and sorafenib was tested using a patient-derived tumor xenograft (PDTX) model. RESULTS: SCD1 overexpression was found in HCC, which was associated with shorter disease-free survival (p = 0.008, log rank test). SCD1 was found to regulate the populations of liver T-ICs; while its suppression by a SCD1 inhibitor suppressed liver T-ICs and sorafenib resistance. Interestingly, SCD1 was markedly upregulated in our established sorafenib-resistant PDTX model, and its overexpression predicts the clinical response of HCC patients to sorafenib treatment. Suppression of SCD1 forces liver T-ICs to differentiate via ER stress-induced unfolded protein response, resulting in an enhanced sensitivity to sorafenib. The PDTX#1 model, combined with sorafenib treatment and a novel SCD1 inhibitor (SSI-4), showed a maximal growth suppressive effect. CONCLUSIONS: SCD1-mediated ER stress regulates liver T-ICs and sorafenib sensitivity. Targeting SCD1 alone or in combination with sorafenib might be a novel personalized medicine against HCC. Lay summary: In this study, SCD1 was found to play a critical role in regulating liver tumor-initiating cells and sorafenib resistance through the regulation of ER stress-mediated differentiation. Targeting SCD1 in combination with sorafenib may be a novel therapeutic strategy against liver cancer.

Down-regulation of TIMP2 by HIF-1α/miR-210/HIF-3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma.

UNLABELLED: Cancer metastasis is a multistep process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling, which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases (TIMP) 2 in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly down-regulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.8%; 23 of 55) in human HCCs and was significantly associated with liver invasion and poorer survival outcomes of HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Using an orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line, MHCC-97L, significantly reduced HCC progression as well as pulmonary metastasis. Mechanistically, TIMP2 suppression, in a hypoxic environment, was induced through a regulatory feedback circuit consisting of hypoxia-inducible factor (HIF) 1 alpha, microRNA-210 (miR-210), and HIF-3α. CONCLUSION: TIMP2 is frequently down-regulated in human HCCs and its down-regulation is associated with aggressive tumor behavior and poorer patient outcome. Its suppression is under the regulation of a novel feedback circuit consisting of HIF-1α/miR-210/HIF-3α. TIMP2 is an important regulator of ECM degradation and HCC metastasis. (Hepatology 2016;64:473-487).

Hypoxia induces myeloid-derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C-C motif) ligand 26.

UNLABELLED: A population of stromal cells, myeloid-derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia-induced MDSC infiltration is dependent on hypoxia-inducible factors. We further found that hypoxia-inducible factors activate the transcription of chemokine (C-C motif) ligand 26 in cancer cells to recruit chemokine (C-X3-C motif) receptor 1-expressing MDSCs to the primary tumor. Knockdown of chemokine (C-C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C-C motif) ligand 26 production in cancer cells by the hypoxia-inducible factor inhibitor digoxin or blockade of chemokine (C-X3-C motif) receptor 1 in MDSCs by chemokine (C-X3-C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. CONCLUSION: This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797-813).

Lysyl oxidase-like 2 is critical to tumor microenvironment and metastatic niche formation in hepatocellular carcinoma.

UNLABELLED: Poor prognosis of cancers, including hepatocellular carcinoma (HCC), is mainly associated with metastasis; however, the underlying mechanisms remain poorly understood. This article investigates the role of lysyl oxidase-like 2 (LOXL-2) in the biology of HCC metastasis. First, we showed that HCC metastasis relies on a collagen-modifying enzyme, LOXL2, which was significantly overexpressed in tumorous tissues and sera of HCC patients, indicating that LOXL2 may be a good diagnostic marker for HCC patients. Second, we delineated a complex, interlinked signaling network that involves multiple regulators, including hypoxia, transforming growth factor beta (TGF-ß), and microRNAs (miRNAs), converging to control the expression of LOXL2. We found not only that LOXL2 was regulated by hypoxia/hypoxia-inducible factor 1 alpha (HIF-1α), but also that TGF-ß activated LOXL2 transcription through mothers against decapentaplegic homolog 4 (Smad4), whereas two frequently underexpressed miRNA families, miR-26 and miR-29, cooperatively suppressed LOXL2 transcription through interacting with the 3' untranslated region of LOXL2. Third, we demonstrated the imperative roles of LOXL2 in modifying the extracellular matrix components in the tumor microenvironment and metastatic niche of HCC. LOXL2 promoted intrahepatic metastasis by increasing tissue stiffness, thereby enhancing the cytoskeletal reorganization of HCC cells. Furthermore, LOXL2 facilitated extrahepatic metastasis by enhancing recruitment of bone-marrow-derived cells to the metastatic site. CONCLUSION: These findings integrate the clinical relevance, molecular regulation, and functional implications of LOXL2 in HCC metastasis.

Epithelioid angiomyolipoma of the liver: a clinicopathologic study of 5 cases.

This study aimed to study the clinicopathologic characteristics of epithelioid angiomyolipoma, a variant of angiomyolipoma (AML) in the liver; and to discuss the diagnostic challenges. Five cases of primary liver epithelioid AML were retrieved from our archives from January 2003 to October 2012. The clinicopathologic features of each case were retrospectively reviewed. All 5 patients were female, with age ranging from 36 to 70 years (median, 41 years). The size of the tumor ranged from 1.2 to 25 cm. Histologically, the tumor comprised polygonal cells with granular eosinophilic cytoplasm and accompanied by immunohistochemical expression of HMB-45 ± Melan-A. Variations in growth pattern and cytology were observed. Estrogen receptor was negative in all 5 cases. None showed cytologic atypia, coagulative necrosis, increased mitotic count, or vascular invasion. Epithelioid AML is an uncommon primary liver tumor with a female predominance. The size of the tumor can be variable. This tumor might impose diagnostic difficulty both clinically and histologically. Immunohistochemical staining with melanocytic markers is a promising means to confirm the pathologic diagnosis. A careful assessment of aggressive histologic features is recommended to stratify the risk of aggressive behavior of this tumor.

Sex-specific analysis of microRNA profiles in HBV-associated cirrhosis by small RNA-sequencing.

Liver cirrhosis represents an advanced stage of chronic liver disease and is associated with significant morbidity, mortality, and risk of cancer development. While sex disparity of liver diseases has been observed, understanding at a genetic level awaits more thorough investigation. In this study, we performed a sex-specific analysis of the microRNA (miR) profiles in hepatitis B virus (HBV)-associated cirrhosis by small RNA-sequencing using clinical tissue samples. Potential associated signaling pathways, downstream gene targets, and upstream regulators were highlighted by computational prediction analyses based on the differentially expressed miRs (DEmiRs). From our results, deregulation of miRs in cirrhosis showed a marked difference between males and females by the degree and pattern. Sixty-five (64 up-regulated, 1 down-regulated) and 12 (6 up-regulated, 6 down-regulated) DEmiRs were found in males and females, respectively, when compared with their respective control group. A number of DEmiRs were only observed in one sex but not the other. In addition, 26 DEmiRs were identified between cirrhosis female and cirrhosis male groups. Fatty acid biosynthesis pathway, extracellular matrix-receptor interaction, p53 signaling, Hippo signaling, tumor necrosis factor signaling, the forkhead box O signaling, as well as gene targets ribosomal protein S27 like, methyl CpG binding protein 2, and estrogen receptor 1, may contribute to the pathogenesis and biological behavior of cirrhosis in a sex-specific manner. Analysis of the Cancer Genome Atlas data set suggested a role of sex-specific DEmiRs in multistep hepatocarcinogenesis. Conclusion: Our findings illustrate that miR profiles in HBV-associated cirrhosis are distinct between the males and females and suggest a potential role of sex-specific biomarkers and molecular mechanisms in disease development and progression.

Clinicopathological and prognostic significance of serum and tissue Dickkopf-1 levels in human hepatocellular carcinoma.

BACKGROUND: Although Dickkopf-1 (DKK1) is known to be a negative regulator of the Wnt/ß-catenin pathway, it has been recently found to be upregulated in cancers. AIMS: We investigated the clinical and prognostic significance of both serum and transcript DKK1 and its functional roles in human hepatocellular carcinoma (HCC). METHODS: We evaluated the expression level of DKK1 in both tissue and serum samples from patients with HCC using GeneChip microarray and real-time-quantitative PCR and sandwich ELISA system respectively. The clinicopathological and prognostic significance of serum and tissue DKK1 levels was examined. Functional characterization of DKK1 with regard to cell migration, invasion and tumour growth was performed. RESULTS: Both DKK1 transcript and serum protein were upregulated in a stepwise manner in human HCCs. Its transcript levels were associated with more aggressive tumour behaviour, in terms of venous invasion (P = 0.003), advanced tumour stage (P = 0.003). DKK1 transcript correlated with shorter overall (P = 0.006) and disease-free survival (P = 0.012), and higher serum DKK1 levels correlated with shorter disease-free survival (P = 0.046). Knockdown of DKK1 significantly reduced both migratory and invasive abilities of HCC cells, whereas overexpression of DKK1 enhanced the tumour formation efficiency and tumour growth in vivo. CONCLUSIONS: Serum and tissue DKK1 levels increased in a stepwise manner in multistep hepatocarcinogenesis and had prognostic significance. DKK1 plays a functional role in cell migration, invasion and tumour growth.

Hepatocellular tumors: immunohistochemical analyses for classification and prognostication.

Following the classification of hepatocellular nodules by the International Working Party in 1995 and further elaboration by the International Consensus Group for Hepatocellular Neoplasia in 2009, entities under the spectrum of hepatocellular nodules have been better characterized. Research work hence has been done to answer questions such as distinguishing high-grade dysplastic nodules from early hepatocellular carcinoma (HCC), delineating the tumor cell origin of HCC, identifying its prognostic markers, and subtyping hepatocellular adenomas. As a result, a copious amount of data at immunohistochemical and molecular levels has emerged. A panel of immunohistochemical markers including glypican-3, heat shock protein 70 and glutamine synthetase has been found to be of use in the diagnosis of small, well differentiated hepatocellular tumors and particularly of HCC. The use of liver fatty acid binding protein (L-FABP), ß-catenin, glutamine synthetase, serum amyloid protein and C-reactive protein is found to be helpful in the subtyping of hepatocellular adenomas. The role of tissue biomarkers for prognostication in HCC and the use of biomarkers in subclassifying HCC based on tumor cell origin are also discussed.

An Immunohistochemical Study of ß-catenin Expression and Immune Cell Population in Metastatic Carcinoma to the Liver.

The liver is the commonest site of cancer metastasis. In this study, we asked whether the immune tumor microenvironment in liver metastases was governed by the ß-catenin activation status of the tumor. To this end, we analyzed CD8 and FoxP3 immunohistochemical expression against ß-catenin expression status of the tumor in a cohort of 52 liver samples with metastatic carcinoma. The results showed that colorectal primary constituted the largest proportion of metastatic carcinoma showing ß-catenin overexpression. Intra-tumoral CD8 count was lower and FoxP3 count was higher when compared with the non-tumoral liver parenchyma. ß-catenin overexpression was associated with a lower CD8 count in the tumor region (p = 0.003). In summary, our findings are in support of an altered immune tumor microenvironment vs. the non-tumor liver tissues in the metastatic site. Suppression of CD8 count was associated with activated Wnt/ß-catenin signaling in the metastatic tumor.

A Clinicopathological Study of Young-onset Hepatocellular Carcinoma.

BACKGROUND/AIM: The aim of this study was to describe the clinicopathological features of hepatocellular carcinoma (HCC) diagnosed at 40 years of age or below. MATERIALS AND METHODS: Expression of CK19, Glypican-3 and ß-catenin was assessed in clinical samples by immunohistochemistry (IHC). IHC expression was correlated with clinicopathological parameters. Hotspot mutations in TP53 gene were analyzed by sequencing. RESULTS: Thirty-six cases were included with a male to female ratio of 3:1. Eighty percent of cases were associated with chronic hepatitis B infection. CK19 and GPC3 were expressed in 61% and 56% of cases, respectively. Only one case demonstrated ß-catenin over-expression. TP53 hotspot mutation was identified in 4 cases. Number of tumor nodules, vascular invasion, and preoperative serum AFP level were associated with prognosis. CONCLUSION: A higher CK19 expression rate was observed in our young-onset HCC cohort, whereas ß-catenin pathway activation and TP53 gene mutation events were less frequent. Conventional clinicopathological parameters remain predictors of survival.

Liver allograft biopsies with histological cholestasis: a clinicopathological study of 254 cases from a single centre.

AIMS: Liver allograft biopsy is important in the management of liver transplant (LT) recipients. Cholestasis is an indicator of liver dysfunction, and histological evidence of cholestasis can be observed in a wide range of pathological entities in the post-LT setting. In this study, we describe the clinicopathological features and significance of liver allograft biopsies with histological cholestasis over 11 years in our centre. METHODS: Liver allograft biopsies performed in Queen Mary Hospital, Hong Kong from 2004 to 2014 showing histological cholestasis were retrieved from the pathology archive. Clinical and pathological data were retrospectively reviewed and analyzed. RESULTS: Among the 254 biopsies from 167 patients, large duct obstruction (LDO) and acute cellular rejection (ACR) were the two main aetiologies associated with cholestasis. There was a decrease in sepsis as a cause over the study duration. In cases showing cholestasis at 6 months or more after LT, LDO was more common than ACR. Over half (61%) of the 254 biopsies showed mild cholestasis. Severe panacinar cholestasis was more often observed in LDO. Mild cholestasis was most commonly observed regardless of the severity of ACR. Severe cholestasis was associated with poorer 1-year and 3-year graft survival and patient survival, as well as higher 3-month and 6-month post-biopsy mortality. CONCLUSION: Histological cholestasis and its severity in liver allograft biopsies has clinical and prognostic significance. Our study summarizes our previous experience and provides further insights into the management of post-LT patients.