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Background: This study aimed to evaluate the role of plasma microRNA panel (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) for prediction and surveillance of early tumor recurrence in hepatocellular carcinoma (HCC) patients who had undergone liver transplantation (LT). Methods: The expression of plasma microRNA panel was assayed in 193 HCC patients (training cohort, n =151; validation cohort, n = 42). Sensitivity and specificity for detecting post-transplant HCC recurrence, and the relationship of microRNA panel expression with clinical characteristics were analyzed accordingly. The prognostic value of microRNA panel was compared with that of AFP (alpha-fetoprotein) and DCP (Des-gamma-carboxyprothrombin). Cox regression analyses were used to evaluate independent prognostic factors. Results: In the training cohort, the rate of positive plasma microRNA panel status at 7-14 days after LT (late phase; 44.2%) decreased than that before (76.2%, P < 0.001) and 1-6 days after LT (early phase; 78.5%, P < 0.001). At late phase, positive microRNA panel status correlated with higher early tumor recurrence rate (one year after LT) than negative status (45.9% vs 10.7%; P < 0.001). Patients with persistent positive microRNA panel status both before and after LT had the highest early tumor recurrence rate in this cohort (54.9%, P < 0.001). The results were consistent in the validation cohort. Cox regression analysis found that positive plasma microRNA panel status at late phase was the only independent risk factor for early recurrence (HR: 4.903, 95% CI = 2.195 - 10.951; P < 0.001). Dynamic monitoring demonstrated plasma microRNA panel status changed from negative to positive earlier than AFP and DCP upon recurrence, and the median time between positivity of plasma microRNA and imaging evidence of recurrence was 2.4 (0.5-10.0) months. Conclusions: Plasma microRNA panel could be a noninvasive biomarker for prediction and surveillance of early tumor recurrence in HCC patients who have undergone LT.

BACKGROUND & AIMS Liver transplantation (LTx) is one of the most effective treatments for hepatocellular carcinoma (HCC); however, tumour recurrence after LTx often leads to poor outcomes. This study investigated the value of circulating tumour cells (CTCs) as a predictor of recurrence following LTx in patients with HCC. METHODS This analysis included 193 patients with HCC who underwent LTx at our institute and accepted pre- and post-operative CTC detection; 38 were selected for serial CTC monitoring. The predictive value of CTCs for tumour recurrence in patients with HCC following LTx was evaluated. Single-cell whole genome sequencing was used to characterize CTCs. RESULTS Overall, the CTC burden decreased after LTx (P

Background Prognosis of hepatocellular carcinoma (HCC) remains poor despite significant recent improvement in therapy. Recent studies have reported that transglutaminase 3 (TGM3) plays an important role in several human cancer types. However, the role of TGM3 in HCC have not been previously elucidated. Methods We evaluated the role of TGM3 in regulating HCC cell proliferation, migration, and invasion. We also investigated the prognostic significance of TGM3 in an HCC cohort. Finally, we explored the signalling pathways that TGM3 regulates in HCC. Results We identified TGM3 to be overexpressed in HCC compared to normal tissues. Higher expression of TGM3 predicts poor prognosis in HCC patients. TGM3 knockdown led to decreased HCC cell proliferation, invasion, and xenograft tumour growth. TGM3 depletion inhibited AKT, extracellular signal–regulated kinase (ERK), p65, and glycogen synthase kinase 3β (GSK3β)/β-catenin activation, but promoted levels of cleaved caspase 3. Moreover, TGM3 knockdown cells had increased E-cadherin levels and decreased vimentin levels, suggesting that TGM3 contributes to epithelial–mesenchymal transition (EMT) in HCC. Conclusion Our results suggest that TGM3 controls multiple oncogenic pathways in HCC, thereby contributing to increased cell proliferation and EMT, and TGM3 potentially enhances HCC metastasis. TGM3 may serve as a novel therapeutic target in HCC.

BackgroundIt is controversial whether adjuvant treatment could be recommended for hepatocellular carcinoma (HCC) after curative hepatectomy. Thus, we performed a network meta-analysis (NMA) to assess adjuvant treatment’s benefit and determine the optimal adjuvant regimen.MethodsWe systematically searched PubMed, Embase, and Cochrane Library for randomized controlled trials comparing adjuvant therapy versus no active treatment after curative hepatectomy among patients with HCC. Pooled data on recurrence and overall survival (OS) were analyzed within pairwise meta-analysis and NMA.ResultsTwenty-three eligible trials (3,940 patients) reporting eight treatments were included. The direct meta-analysis showed that adjuvant therapy prevented the recurrence (OR = 0.65; 95% CI: 0.55, 0.77; P = 0.177; I2 = 21.7%) and contributed to OS (HR = 0.63; 95% CI: 0.54, 0.73; P = 0.087; I2 = 31.1%) in comparison to the observation. In the NMA, internal radiotherapy (IRT; OR = 0.55; 95% CI: 0.39, 0.77; SUCRA = 87.7%) followed by hepatic artery infusion chemotherapy (HAIC; OR = 0.6; 95% CI: 0.36, 0.97; SUCRA = 77.8%), and HAIC (HR = 0.44; 95% CI: 0.21, 0.87; SUCRA = 82.6%) followed by IRT (HR 0.54; 95% CI:0.36, 0.81; SUCRA = 69.7%) were ranked superior to other treatments in terms of preventing recurrence and providing survival benefit, respectively.ConclusionsThe addition of adjuvant therapy lowers the risk of recurrence and provide survival benefit after surgical resection for HCC. HAIC and IRT are likely to be the two most effective adjuvant regimens.Systematic Review Registrationhttps://inplasy.com/inplasy-2020-11-0039/.

Background & Aims Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. Methods We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. Results We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. Conclusions Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. Lay summary We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

ObjectiveThe lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma (HCC). We sought to develop a non-invasive diagnostic approach using circulating cell-free DNA (cfDNA) for the early detection of HCC.DesignApplying the 5hmC-Seal technique, we obtained genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA samples from 2554 Chinese subjects: 1204 patients with HCC, 392 patients with chronic hepatitis B virus infection (CHB) or liver cirrhosis (LC) and 958 healthy individuals and patients with benign liver lesions. A diagnostic model for early HCC was developed through case-control analyses using the elastic net regularisation for feature selection.ResultsThe 5hmC-Seal data from patients with HCC showed a genome-wide distribution enriched with liver-derived enhancer marks. We developed a 32-gene diagnostic model that accurately distinguished early HCC (stage 0/A) based on the Barcelona Clinic Liver Cancer staging system from non-HCC (validation set: area under curve (AUC)=88.4%; (95% CI 85.8% to 91.1%)), showing superior performance over α-fetoprotein (AFP). Besides detecting patients with early stage or small tumours (eg, ≤2.0 cm) from non-HCC, the 5hmC model showed high capacity for distinguishing early HCC from high risk subjects with CHB or LC history (validation set: AUC=84.6%; (95% CI 80.6% to 88.7%)), also significantly outperforming AFP. Furthermore, the 5hmC diagnostic model appeared to be independent from potential confounders (eg, smoking/alcohol intake history).ConclusionWe have developed and validated a non-invasive approach with clinical application potential for the early detection of HCC that are still surgically resectable in high risk individuals.

Objective Differentiation‐inducing therapy for tumors is a strategy that aims to induce the differentiation and maturation of cancer stem cells (CSCs). The differentiation‐inducing capacity of arsenic trioxide (ATO) in hepatocellular carcinoma (HCC) and the underlying mechanism were previously unknown. Methods In the present study, we explored the ATO‐induced differentiation of CSCs in HCC by detecting the expression of CSC‐related markers and tumorigenicity variation in vivo and in vitro. We developed a combined chemotherapeutic approach to HCC by characterizing the effects of combinatorial treatment with 5‐fluorouracil (5‐FU)/cisplatin and ATO in vitro and in patient‐derived xenograft models. Changes in gene expression patterns were investigated by gene microarray analysis. Results ATO effectively induced differentiation of CSCs by downregulation of CSC‐related genes and suppression of tumorigenicity capability. Combinatorial treatment with ATO and 5‐FU/cisplatin significantly enhanced therapeutic effects in HCC cells compared with the treatment with 5‐FU/cisplatin alone. Synergistic inhibition of the LIF/JAK1/STAT3 and NF‐kB signaling pathways by ATO and 5‐FU/cisplatin is a potential molecular mechanism underlying the differentiation effect. Conclusions ATO induced the differentiation of HCC CSCs and potentiated the cytotoxic effects of 5‐FU/cisplatin through synergistic inhibition of the LIF/JAK1/STAT3 and NF‐kB signaling pathways. These results offer new insights for the clinical treatment of HCC., Arsenic trioxide induces differentiation of cancer stem cells in hepatocellular carcinoma through inhibition of LIF/JAK1/STAT3 and NF‐kB signaling pathways synergistically

Background and aims There is growing evidence that single-stranded, circular RNA (circRNA) plays a key role in the development of certain cancers, including hepatocellular carcinoma (HCC). It is less clear, however, what role circRNA plays in HCC metastasis. Approach and results In this study, through circRNA sequencing, we identified a circRNA: circASAP1 (a circRNA derived from exons 2 and 3 of the ASAP1 gene, hsa_circ_0085616), which is associated with pulmonary metastasis after curative resection in patients with HCC. CircASAP1 was overexpressed in HCC cell lines with high metastatic potential and in metastatic HCCs. In vitro, circASAP1 promoted cell proliferation, colony formation, migration, and invasion, and in vivo, it enhanced tumor growth and pulmonary metastasis. Mechanism studies showed that circASAP1 acts as a competing endogenous RNA for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), both of which are tumor suppressors in HCC. We found that mitogen-activated protein kinase (MAPK) 1 and colony stimulating factor (CSF)-1 were direct common targets for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), which were regulated by circASAP1. CircASAP1 promotes HCC cell proliferation and invasion by regulating miR-326/miR-532-5p-MAPK1 signaling and, furthermore, mediates tumor-associated macrophage infiltration by regulating the miR-326/miR-532-5p-CSF-1 pathway. Clinical HCC samples exhibited a positive correlation between circASAP1 expression and levels of CSF-1, MAPK1, and CD68+ tumor-associated macrophages, all of which were predictive of patient outcomes. Conclusion We identified circASAP1 as a key regulator of HCC metastasis that acts on miR-326/miR-532-5p-MAPK1/CSF-1 signaling and serves as a prognostic predictor in patients with HCC.

Background. Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer-related mortality in China with increasing incidence. This study is designed to explore early genetic changes implicated in HCC tumorigenesis and progression by whole-exome sequencing. Methods. We firstly sequenced the whole exomes of 5 paired hepatitis B virus-related early-stage HCC and peripheral blood samples, followed by gene ontological analysis and pathway analysis of the single-nucleotide variants discovered. Then, the mutations of high frequency were further confirmed by Sanger sequencing. Results. We identified a mutational signature of dominant T:A>A:T transversion in early HCC and significantly enriched pathways including ECM-receptor interaction, axon guidance, and focal adhesion and enriched biological processes containing cell adhesion, axon guidance, and regulation of pH. Eight genes, including MUC16, UNC79, USH2A, DNAH17, PTPN13, TENM4, PCLO, and PDE1C, were frequently mutated. Conclusions. This study reveals a mutational profile and a distinct mutation signature of T:A>A:T transversion in early-stage HCC with HBV infection, which will enrich our understanding of genetic characteristics of the early-stage HCC.

The semaphorins were originally identified as having roles as guidance cues during neural development. Class 3 semaphorins are involved in cancer progression. However, the roles of class 3 semaphorins in hepatocellular carcinoma (HCC) are unknown. We examined the expression levels of class 3 semaphorins in HCC cell lines with different metastatic potential and in carcinoma tissue samples. The results indicated that Semaphorin 3A expression was up-regulated in metastatic cell lines and in samples from patients with tumor recurrence. Cell functional studies revealed that Semaphorin 3A promoted HCC cell proliferation, migration, and invasion. Animal studies indicated that Semaphorin 3A overexpression enhanced tumor growth and lung metastasis. Semaphorin 3A also acted as a chemoattractant involved in direct recruitment of macrophages in vitro, and facilitated tumor-associated macrophage (TAM) infiltration in vivo. Multivariate analysis revealed that Semaphorin 3A expression alone, or combined with the number of TAMs, can be an independent predictor for overall survival time and time to recurrence. Overall, the results suggested that Semaphorin 3A increased TAM infiltration and promoted HCC progression. Semaphorin 3A expression alone, or combined with the number of TAMs, is a new prognostic factor and potential target for the treatment of HCC.

Background Survival after resection of hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) still remains poor. Apatinib, a vascular endothelial cell growth factor receptor 2 inhibitor, has been shown to be safe and effective in patients with advanced HCC, so in the present study its efficacy and safety in the adjuvant setting was explored. Methods In this single-center, open-label phase II trial, the patients received apatinib (500 mg/day) until they experienced disease recurrence or intolerable toxicity. The primary endpoint was recurrence-free survival (RFS); the secondary endpoints included overall survival (OS) and safety. Results From a total of 49 patients who were screened between August 2017 and December 2018, 30 study participants received apatinib. According to the Liver Cancer Study Group of Japan classification of PVTT, there were 7, 11, and 12 participants with Vp1, Vp2, and Vp3, respectively. The median duration of treatment was 4.8 months [interquartile range (IQR): 2.0-8.8], and the median dose of apatinib was 339.7 mg/day (IQR: 267.7-500 mg/day). The median follow-up was 14.3 months (IQR: 12.3-19.3). The median RFS was 7.6 months [95% confidence interval (CI): 5.7-9.5 months]. The 1-year RFS rate and the 1-year OS rate were 36.1% and 93.3%, respectively. A total of 29 (96.7%) patients experienced adverse events, and 14 (46.7%) had grade 3 or 4 adverse events. No treatment-related deaths occurred. Conclusions Apatinib was well tolerated in patients after resection of HCC with PVTT. The median RFS in this group was improved compared with that previously reported. Trial registration No.: NCT03261791 (ClinicalTrials.gov).

514 Background: Adjuvant therapy for hepatocellular carcinoma (HCC) is an unmet need. Apatinib, a VEGFR2 inhibitor, showed antitumor activity and tolerable toxicity for advanced HCC in a phase 2 study. We were aiming to explore the safety and efficacy of apatinib in adjuvant settings after resection of HCC with portal vein tumor thrombosis (PVTT). Methods: This is a single-center single-arm phase 2 study. The key inclusion criteria were: (1) pathologically confirmed HCC; (2) underwent liver resection with curative intention within 4-6 wk before recruitment; (3) PVTT assessed by preoperative imaging or intraoperative findings. Eligible pts received apatinib at 500 mg/day for a maximum of 12 mo until unacceptable toxicity or tumor recurrence. The primary outcome was recurrence-free survival (RFS) defined as the interval between surgery and the diagnosis of tumor recurrence or death from any causes whichever comes first. The secondary outcome is overall survival (OS) and treatment safety. Results: From Aug 17, 2017 to Dec 18, 2018, 49 pts were screened, and 30 pts were recruited. PVTT were classified as Vp1 (n = 7), Vp2 (n = 11), and Vp3 (n = 12) according to the LCSGJ classification. As the data cutoff on Aug 22, 2019, 4 pts are still on treatment. The median follow-up was 14.3 mo (IQR 12.3-19.3). Median duration of treatment was 4.8 mo (IQR 2.0–8.8). 20 recurrence and 2 death occurred including one death without recurrence. The mRFS was 7.6 mo (95% CI, 3.7-11.5), and the 1-year RFS rate was 36.1%. The mOS was not reached, and 1-year OS rate was 93.3% (standard error, 4.6%). Treatment-related adverse events occurred in 29 pts (96.7%). Grade 3 or 4 adverse events were reported in 14 pts (46.7%) (Table). Dose modification due to adverse events was recorded in 23 pts (76.7%). Conclusions: Apatinib is tolerant in most pts after resection for HCC with PVTT. A controlled study is warranted to prove its efficacy on tumor recurrence. Clinical trial information: NCT03261791. [Table: see text]

Liver cancers are highly heterogeneous with poor prognosis and drug response. A better understanding between genetic alterations and drug responses would facilitate precision treatment for liver cancers. To characterize the landscape of pharmacogenomic interactions in liver cancers, we developed a protocol to establish human liver cancer cell models at a success rate of around 50% and generated the Liver Cancer Model Repository (LIMORE) with 81 cell models. LIMORE represented genomic and transcriptomic heterogeneity of primary cancers. Interrogation of the pharmacogenomic landscape of LIMORE discovered unexplored gene-drug associations, including synthetic lethalities to prevalent alterations in liver cancers. Moreover, predictive biomarker candidates were suggested for the selection of sorafenib-responding patients. LIMORE provides a rich resource facilitating drug discovery in liver cancers.

The lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma (HCC), the second-most common cause of cancer deaths worldwide. We sought to develop a clinically convenient and minimally-invasive approach that can be deployed at scale for the sensitive, specific, and highly reliable diagnosis of HCC, and to evaluate the potential prognostic value of this approach. The study cohort comprised of 2,728 subjects, including HCC patients (n = 1,208), controls (n = 965) (572 healthy individuals and 393 patients with benign lesions), as well as patients with chronic hepatitis B infection (CHB) (n =291), liver cirrhosis (LC) (n = 110), and cholangiocarcinoma (CCC) (n = 154), was recruited from three major liver cancer hospitals in Shanghai, China, from July 2016 to November 2017. Circulating cell-free DNA (cfDNA) were collected from plasma samples from these individuals before surgery or any radical treatment. Applying our 5hmC-Seal technique, the summarized 5-hydroxymethylcytosine (5hmC) profiles in cfDNA were obtained. Molecular annotation analysis suggested that the profiled 5hmC loci in cfDNA were enriched with liver tissue-derived regulatory markers (e.g., H3K4me1). We showed that a weighted diagnostic score (wd-score) based on 117 genes detected using the summarized 5hmC profiles in cfDNA accurately distinguished HCC patients from controls (AUC = 95.1%; 95% CI, 93.6-96.5%) in the validation set, markedly outperformed α-fetoprotein (AFP) with superior sensitivity. The wd-scores, which not only detected early BCLC stages (e.g., Stage 0: AUC = 96.2%; 95% CI,94.1-98.4%) and small tumors (e.g., < 2 cm: AUC = 95.7%; 95% CI: 93.6-97.7%), also showed high capacity for distinguishing HCC from non-cancer patients with CHB/LC (AUC = 80.2%; 95% CI, 75.8-84.6%). Moreover, the prognostic value of 5hmC markers in cfDNA was evaluated for HCC recurrence, showing that a weighted prognostic score (wp-score) based on 16 marker genes predicted the recurrence risk (HR = 6.67; 95% CI, 2.81-15.82, p < 0.0001) in 555 patients who have been followed up after surgery. In conclusion, we have developed and validated a robust 5hmC-based diagnostic model that can be applied routinely with clinically feasible amount of cfDNA (e.g., from ~2-5 mL of plasma). Applying this new approach in the clinic could significantly improve the clinical outcomes of HCC patients, for example by early detection of those patients with surgically resectable tumors or as a convenient disease surveillance tool for recurrence.

Increasing numbers of evidences have demonstrated that microRNAs (miRNAs) are implicated in metastasis and progression of hepatocellular carcinoma (HCC). However, their detailed expression levels and actual functions in HCCs have not been fully clarified yet. Results from our recent study revealed that some miRNAs were particularly related to metastasis of HCCs. As one of these newly found miRNAs, miR-501-3p showed to highly involve into metastatic process of HCCs. Here we reported that the expression of miR-501-3p was decreased in both metastatic HCC cell lines and tissue samples from HCC patients with recurrence and metastasis. Downregulation of miR-501-3p correlated with tumor progression and poor prognosis in the HCC patients. Results of functional analyses revealed that overexpression of miR-501-3p in HCCLM3 cancer cells inhibited their proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT), while miR-501-3p loss in PLC/PRF/5 cancer cells facilitated all these cellular activities. In addition, Lin-7 homolog A (LIN7A) was directly targeted by miR-501-3p to mediate the suppression effects on metastasis in HCC cells. miR-501-3p suppresses metastasis and progression of HCCs through targeting LIN7A. This finding suggests that miR-501-3p could be used as a potential prognostic predictor as well as a potential therapeutic tool for HCC therapies.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality and carries a dismal prognosis. The present study aimed to identify the tumour-suppressive role and clinical implications of PTPN13 in HCC progression. We tested the effects of PTPN13 expression in proliferation, invasion, epithelial-mesenchymal transition and associated pathways in HCC cell lines in vitro. Furthermore, its clinical relevance was evaluated in a tissue microarray analysis of samples from 282 HCC patients. Various HCC cell lines expressed relatively low PTPN13 protein levels in vitro. PTPN13 overexpression significantly inhibited the progression of HCC cells, possibly by inhibiting epithelial-mesenchymal transition through inactivation of the EGFR/ERK signalling pathway. Tissue microarray analysis revealed that high PTPN13 expression was correlated with a favourable prognosis in postoperative HCC patients. This study demonstrated the tumour suppressor, PTPN13, as an alternative therapeutic target for HCC.

The clinical significance of circulating cell-free DNA (cfDNA) integrity as diagnostic and surveillance biomarker in hepatocellular carcinoma (HCC) was investigated and compared to that of alpha fetoprotein (AFP). Liver cancer patients had lower cfDNA integrity than those with benign diseases (P = 0.0167) and healthy individuals (P = 0.0025). Patients with HCC and non-HCC liver cancers (P = 0.7356), and patients with benign diseases and healthy individuals (P = 0.9138) had comparable cfDNA integrity respectively. cfDNA integrity increased after hepatectomy in cancer patients (P = 0.0003). The AUCs for detecting HCC by cfDNA integrity and AFP were 0.705 (P = 0.005) and 0.605 (P = 0.156), respectively. We found cfDNA integrity decreased in HCC patients and has the potential as promising biomarker for HCC diagnosis and treatment surveillance.

Previous studies have indicated that complex interactions among viral, environmental and genetic factors lead to hepatocellular carcinoma (HCC). To identify susceptibility alleles for hepatitis B virus (HBV)-related HCC, the present study conducted a pilot two-phase genome-wide association study (GWAS) in 660 Han Chinese individuals. In phase 1, a total of 500,447 single-nucleotide polymorphisms (SNPs) were genotyped in 50 HCC cases and 50 controls using Affymetrix GeneChip 500k Array Set. In phase 2, 1,152 SNPs were selected from phase 1 and genotyped in 282 cases and 278 controls using the Illumina GoldenGate platform. The prior probability of HCC in control subjects was assigned at 0.01, and false-positive report probability (FPRP) was utilized to evaluate the statistical significance. In phase 1, one SNP (rs2212522) showed a significant association with HCC (Pallele=5.23×10−8; ORallele=4.96; 95% CI, 2.72–9.03). In phase 2, among 27 SNPs with unadjusted Pallele

Studies have highlighted important features of the nucleocytoplasmic transport of mRNAs and proteins. Nuclear RNA export factor 3 (NXF3) is a member of the nuclear RNA export factor family that plays a role in mediating the export of cellular mRNA from the nucleus to the cytoplasm for translation. However, little is known about the clinical significance of NXF3 in human tumors. To evaluate the prognostic significance of NXF3 in hepatocellular carcinoma (HCC), the expression levels of NXF3 in a cohort of 112 patients with primary HCC who had undergone hepatectomy for histologically confirmed HCC were assessed by immunohistochemistry. It was identified that the expression levels of NXF3 were higher in the primary HCC tissues compared with those in paired peritumoral liver tissues. The overexpression of NXF3 in the HCC tissues was correlated with decreased survival time [hazard ratio (HR) = 1.954, 95% confidence interval (CI) = 1.034–3.695, P=0.039] and earlier tumor recurrence (HR = 2.101, 95% CI = 1.186–3.722, P=0.011) in postoperative patients with HCC. Notably, overexpression of NXF3 was correlated with a poor survival time and increased recurrence following HCC resection in male patients (P=0.020 and P=0.007, respectively) but not in female patients (P=0.916 and P=0.821, respectively). In conclusion, the findings provide evidence that implicates NXF3 as a prospective predictor of HCC prognosis as well as a potential therapeutic target for cancer treatment.

The calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles (DCVs) in neurons and neuroendocrine cells. The role of CAPS1 in cancer biology remains unknown. The purpose of this study was to investigate the role of CAPS1 in hepatocellular carcinoma (HCC). We determined the levels of CAPS1 in eight hepatoma cell lines and 141 HCC specimens. We evaluated the prognostic value of CAPS1 expression and its association with clinical parameters. We investigated the biological consequences of CAPS1 overexpression in two hepatoma cell lines in vitro and in vivo. The results showed that loss of CAPS1 expression in HCC tissues was markedly correlated with aggressive tumor phenotypes, such as high-grade tumor node metastasis (TNM) stage (p = 0.003) and absence of tumor encapsulation (p = 0.016), and was associated with poor overall survival (p = 0.008) and high recurrence (p = 0.015). CAPS1 overexpression inhibited cell proliferation and migration by changing the exocytosis-associated tumor microenvironment in hepatoma cells in vitro. The in vivo study showed that CAPS1 overexpression inhibited xenograft tumor growth. Together, these results identified a previously unrecognized tumor suppressor role for CAPS1 in HCC development.

Purpose: Circulating tumor DNA (ctDNA) is increasingly recognized as liquid biopsy to profile tumor genome. Droplet digital PCR (ddPCR) is a highly sensitive and easily operable platform for mutant detection. Here, we tried to detect ctDNA in hepatocellular carcinoma (HCC) patients using ddPCR. Methods: Studies sequencing the genome of HCCs and COSMIC (Catalogue of Somatic Mutations in Cancer) database were reviewed to identify hotspot mutations. Circulating cell-free DNAs (cfDNAs) extracted from 1 ml preoperative plasma sample were analyzed to detect circulating mutants using ddPCR. The DNAs from matched tumor and adjacent liver tissues or peripheral blood mononuclear cells (PBMCs) were sequenced to identify the origin of circulating mutants. Results: Forty-eight HCC patients were enrolled and four gene loci, TP53 (c.747G>T), CTNNB1 (c.121A>G, c.133T>C), and TERT (c.1-124C>T) were chosen as targets for ddPCR assay. Serial dilution demonstrated the detection limit of ddPCR to be 0.01%. Twenty-seven patients (56.3%, 27/48) were found to have at least one kind of circulating mutants, with the mutant allele frequency ranging from 0.33% to 23.7%. Six patients (22.2%, 6/27) also had matched mutants in tumor tissues while none of the mutants were detected in adjacent liver tissues or PBMCs in all patients, which excluded the nonneoplastic origin of these circulating mutants and qualified them as ctDNA. Conclusions: ctDNA could be readily detected in HCC patients by targeting hotspot mutations using ddPCR and might reflect intratumoral heterogeneity. ctDNA detecting may serve as a promising liquid biopsy in HCC management.

CXCL5 (epithelial neutrophil-activating peptide-78) is a member of a proangiogenic subgroup of the CXC-type chemokine family of small, secreted proteins. Recently, evidence that CXCL5 is involved in carcinogenesis and cancer progression has emerged. To investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC), we examined CXCL5 messenger RNA (mRNA) and protein levels in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. We found that CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to controls. CXCL5 activated the PI3K-Akt and ERK1/2 signaling pathways in HCC cells and promoted proliferation, migration, and invasion. Furthermore, we found that CXCL5 had a direct chemoattractant effect on neutrophils in vitro. In animal studies, the up-regulation of CXCL5 in HCC cells promoted tumor growth, lung metastasis, and intratumoral neutrophil infiltration. Conversely, down-regulation of CXCL5 in HCC cells reduced tumor growth, metastasis, and intratumoral neutrophil infiltration. Immunohistochemical analysis in HCC samples showed that overexpression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for overall survival and cumulative recurrence. Conclusion: CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC. (HEPATOLOGY 2012;56:2242–2254)

An immune function assay has been proposed as a new strategy to monitor immunosuppression after organ transplantation. However, there are limited data regarding its role in liver transplant recipients with hepatocellular carcinoma (HCC). In this study, we sought to determine the utility of this functional assay in assessing the risk of infection, rejection, and tumor recurrence in liver transplant recipients.Immune function was determined by ImmuKnow assay that measures the amount of adenosine triphosphate (ATP) produced by CD4 (+) T cells to monitor the global immune status in 342 whole blood samples from 105 liver transplant recipients. The association between ATP value and post-transplant tumor recurrence was evaluated in 60 HCC patients. The ATP value in predicting tumor recurrence in other independent cohort of 92 recipients with HCC was analyzed prospectively.The mean ATP values of liver transplant recipients with infection (145.2 ± 87.0 ng/ml) or acute rejection (418.9 ± 169.5 ng/ml) were different from those with stable state (286.6 ± 143.9 ng/ml, P 0.05). In recipients with HCC who developed recurrent tumors, the values were significantly lower than those without recurrence (137.8 ± 66.4 vs. 289 ± 133.9 ng/ml, P 0.01); the optimal threshold value to predict post-transplant tumor recurrence was 175 ng/ml. Comparing with the patients in lower immune group (ATP ≤ 175 ng/ml), patients in the higher immune group (ATP 175 ng/ml) experienced significantly better disease-free survival (P 0.01). Multivariate Cox regression analysis showed the ATP value was an independent predictor of HCC recurrence.The immune function assay has the potential to assess the risk of infection and rejection in liver transplantation and to predict post-transplant tumor recurrence in recipients with HCC.

Background A retrospective cohort study was conducted to investigate the effect of interferon-α (IFN-α) therapy after curative resection on survival and recurrence in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Methods Of 568 HBV-related HCC patients who underwent curative resection, 101 patients received postoperative IFN-α therapy (5 million units three times every week for 18 months). Clinicopathological factors were compared between patients with postoperative IFN-α therapy or not. Risk factors for survival, early, and late recurrence (2 years as cut-off) were studied. Results The median follow-up time was 53.3 months. There was no significant difference in clinicopathological factors between the two groups. Patients with postoperative IFN-α therapy had higher overall survival rates (hazards ratio (HR): 0.612, 95% confidence interval (CI): 0.422–0.889, P = 0.010). No significant difference in disease-free survival rates was detected between the two groups (HR: 0.786, 95% CI: 0.597–1.035, P = 0.086). Multivariate analysis revealed that postoperative IFN-α therapy was an independent factor for overall survival (HR: 0.611, 95% CI: 0.421–0.887, P = 0.010) and significantly reduced early recurrence (HR: 0.562, 95% CI: 0.375–0.840, P = 0.005). Conclusions IFN-α therapy after curative resection prevented early recurrence and improved overall survival of patients with HBV-related HCC. J. Surg. Oncol. 2010;102:796–801. © 2010 Wiley-Liss, Inc.

A retrospective cohort study was conducted to identify risk factors for recurrence of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after curative resection. A total of 317 patients who had received curative resection of pathologically proven small HCC (or = 3 cm in diameter) were analyzed to ascertain the factors affecting recurrence. The median follow-up period was 33.7 months. Cumulative recurrence rates at 1, 3, and 5 years after resection were 23.5%, 49.5%, and 65.5%, respectively. Male sex, alpha-fetoprotein (AFP)or = 400 ng/mL, HBV DNA levelor = 4 log(10) copies/mL, prolonged prothrombin time, tumor sizeor = 2 cm, microvascular invasion, absence of capsular formation, moderate/poor tumor differentiation, and absence of postoperative interferon-alpha (IFN-alpha) treatment were associated with increased cumulative risk of HCC recurrence. By multivariate analysis, HBV DNA levelor = 4 log(10) copies/mL (P0.001, hazard ratio (HR) 2.110), AFPor = 400 ng/mL (P = 0.011, HR 1.574), microvascular invasion (P0.001, HR 1.767), and postoperative IFN-alpha treatment (P = 0.022, HR 0.562) remained to be independently associated with HCC recurrence. Those contributing to late recurrence (2 years) were older age and HBV DNA levelor = 4 log(10) copies/mL. Patients with persistent HBV DNA levelor = 4 log(10) copies/mL at resection and follow-up had the highest recurrence risk (P0.001, HR 4.129). HBV DNA levelor = 4 log(10) copies/mL at the time of resection was the most important risk factor for recurrence. Postoperative IFN-alpha treatment significantly decreased the recurrence risk after resection.

The optimum strategy, hepatic resection (HR) or liver transplantation (LT), for treatment of early hepatocellular carcinoma (HCC) associated with liver diseases of Child–Pugh A is far from established. The aim of this study was to compare and determine which strategy is optimal for HCC fulfilling the Milan criteria. Consecutive data were collected in 1,018 HCC patients treated with HR and 89 HCC patients listed for LT (1 drop out for HCC progression) between January of 2003 and December of 2007. The independent prognostic factors identified by multivariate analysis were tumor size-plus-number, microscopic venous invasion, and operation type (LT or HR). When tumor size-plus-number was ≤4 or microscopic venous invasion was absent, there was no significant difference in overall survival (OS) between the LT and HR group. When tumor size-plus-number was >4 or microscopic venous invasion was present, OS was higher in the LT group. Since the pathological microscopic venous invasion was not easily available before operation which is limitation for widespread clinical use, thus in practice, we concluded that, for early HCC associated with Child–Pugh A cirrhosis, when tumor size-plus-number is >4, LT provides the best cure; when it is ≤4, HR remains the initial treatment of choice.

Immunosuppressive therapy after liver transplantation for hepatocellular carcinoma (HCC) is one of the major contributory factors for HCC recurrence and metastasis. Sirolimus, a potent immunosuppressant, has been reported to be an effective inhibitor in a variety of tumors. The present study is designed to explore whether sirolimus could block the growth and metastatic progression of HCC.MHCC97H cells were used as targets to explore the effect of sirolimus on cell cycle progression, apoptosis, proliferation, and its antiangiogenic mechanism. LCI-D20, a highly metastatic model of human HCC in nude mice, was also used as the model tumor to explore the effect of sirolimus on tumor growth and metastatic progression.In vitro, sirolimus induced cell cycle arrest at the G1 checkpoint and blocked proliferation of MHCC97H cells but did not induce apoptosis. In vivo, sirolimus prevented tumor growth and metastatic progression in LCI-D20. Intratumoral microvessel density and circulating levels of VEGF in tumor-bearing mice were also significantly reduced in sirolimus treatment group. Quantitative RT-PCR showed that sirolimus down-regulated the mRNA expression of VEGF and HIF-1a, but not of bFGF, and TGF-b in MHCC97H cells. Furthermore, western blot analysis confirmed that sirolimus also decreased expression of HIF-1a at protein level, in parallel with the down-regulation of the levels of VEGF protein excretion in a time-dependent manner as compared to untreated control cells following anoxia.The immunosuppressive macrolide sirolimus prevents the growth and metastatic progression of HCC, and suppresses VEGF synthesis and secretion by downregulating HIF-1a expression. Sirolimus may be useful for clinical application in patients who received a liver transplant for HCC.

To evaluate the prognostic value of tumor-associated macrophages (TAM), individually or synergistically with CD45RO + memory T cells (T(M)), in hepatocellular carcinoma (HCC) patients following resection.The infiltration of TAM and T(M) was assessed by immunohistochemistry in tissue microarray containing 302 HCC specimens. Correlations between TAM/T(M) infiltration and clinicopathologic features, disease-free survival (DFS) and overall survival (OS) were statistically analyzed.High TAM infiltration was associated with both improved DFS (P = 0.0021) and OS (P = 0.0481). Multivariate analysis identified TAM infiltration as independent prognostic factor for DFS (P = 0.004) and OS (P = 0.049). A second analysis clarified the synergistic effect of TAMT(M) infiltration for DFS (P = 0.004) and OS (P = 0.040).Both TAM infiltration alone and concomitant infiltration of TAMT(M) are associated with improved DFS/OS, suggesting that TAM could protect HCC patients from recurrence/metastasis and prolong survival by distinct mechanisms.

Purpose: Novel therapeutic strategies are needed to prevent the tumor recurrence or metastasis after liver transplantation for hepatocellular carcinoma (HCC). This study was to investigate the effect of rapamycin, alone and in combination with sorafenib, on HCC in vivo. Experimental Design: Xenograft of a highly metastatic human HCC tumor (LCI-D20) was used to evaluate primary tumor growth and lung metastasis after treatment with rapamycin alone or in combination with sorafenib. Tumor cell proliferation was determined by Ki-67 immunostaining. To detect tumor cell apoptosis, the terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling assay was used. Tumor angiogenesis was investigated by using a monoclonal anti-CD31 antibody. A vascular endothelial growth factor ELISA kit was used to measure vascular endothelial growth factor protein levels in the mice serum. Results: Rapamycin, alone and in combination with sorafenib, strongly inhibited primary tumor growth and lung metastases in LCI-D20 model. Furthermore, the combination therapy significantly enhanced the effect of antitumor on primary tumor growth compared with single treatment with either rapamycin (P < 0.001) or sorafenib (P < 0.001). Rapamycin alone inhibited HCC cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Nevertheless, the combination therapy showed a significant inhibition of tumor cell proliferation (P < 0.05). Additionally, the combination therapy also further enhanced suppression of tumor cell angiogenesis compared with rapamycin treatment (P < 0.01). However, the induction of apoptosis in combination therapy group was not significantly higher than in the rapamycin-treated group (P > 0.05). Conclusions: The combination therapy of rapamycin and sorafenib could be a new and promising therapeutic approach to the treatment of HCC and prevention of HCC recurrence after liver transplantation.

MicroRNAs (miRNAs) play a critical role in the regulation of tumor metastasis. The role of these molecules in hepatocellular carcinoma (HCC), however, has not been fully elucidated. In this study, we employed miRNA-sequencing and identified 22 miRNAs involved in HCC metastasis. One of these, miR-28-5p, was downregulated in HCCs. This downregulation correlated with tumor metastasis, recurrence, and poor survival. Biofunctional investigations revealed that miR-28-5p deficiency promoted tumor growth and metastasis in nude mice without altering the in vitro biological characteristics of HCC cells. Through gene expression profiles and bioinformatics analysis, we identified interleukin-34 (IL-34) as a direct target of miR-28-5p, and the effects of miR-28-5p deficiency on HCC growth and metastasis was dependent on IL-34-mediated tumor-associated macrophage (TAMs) infiltration. Moreover, we found that TAMs induced by miR-28-5p-IL-34 signaling inhibit miR-28-5p expression on HCC cells via transforming growth factor-β1 (TGF-β1), resulting in an miR-28-5p-IL-34-macrophage positive feedback loop. In clinical HCC samples, miR-28-5p levels were inversely correlated with IL-34 expression and the number of TAMs. Patients with low miR-28-5p expression, high IL-34 levels, and high numbers of TAMs had a poor prognosis with shorter overall survival (OS) and time to recurrence (TTR). Conclusion: an miR-28-5p-IL-34-macrophage feedback loop modulates HCC metastasis and serves as a novel prognostic factor as well as a therapeutic target for HCC. This article is protected by copyright. All rights reserved.

Clinical response of hepatocellular carcinoma (HCC) to arsenic trioxide (ATO) has been poor. Promyelocytic leukemia protein (PML) is central to ATO treatment efficacy of acute promyelocytic leukemia. We examine impacts of PML expression on the effectiveness of ATO treatment in HCC. We show that increased PML expression predicts longer survival and lower cancer recurrence rates after HCC resection. However, high PML expression dampens the anti-tumor effects of ATO in HCC cells. Gene microarray analysis shows that reduced PML expression significantly down-regulates expression of aldehyde dehydrogenase 3 family member A1 (ALDH3A1). ALDH3A1 depression facilitates accumulation of ATO-induced reactive oxygen species. Chromatin immunoprecipitation analysis and promoter activity assays confirm that PML regulates ALDH3A1 expression through binding to the promoter region of ALDH3A1. Clinically, ATO treatment decreases the disease progression rate in advanced HCC patients with negative PML expression. In conclusion, PML confers a favorable prognosis in HCC patients, but it induces ATO resistance through ALDH3A1 up-regulation in HCC cells. ATO is effective for HCC patients with negative PML expression. Combined with an ALDH3A1 inhibitor, ATO may be efficacious in patients with positive PML expression.

Hepatocellular carcinoma (HCC) is one of the most serious health problems worldwide. As in many other diseases, environment and genetic factors are believed to be involved in the pathogenesis of HCC. Numerous epidemiologic investigations including case–control and cohort studies have suggested the association of glutathione S-transferase (GST) genetic polymorphisms and HCC risk. However, some studies have produced conflicting results. Therefore, we performed an updated meta-analysis to clarify this inconsistency and to establish a comprehensive picture of the association of the polymorphisms of GSTM1 and GSTT1 with HCC susceptibility. We searched PubMed, Embase, ISI Web of Science, and CNKI databases to identify eligible studies meeting the inclusion criteria up to August 30, 2013. Odds ratios (ORs) and 95 % confidence intervals (CIs) were used to assess the strength of association. Finally, there were a total of 33 studies with 4,232 cases and 6,601 controls included in this meta-analysis. In the pooled analysis, significantly increased HCC risks were found for null genotype of GSTM1 (OR = 1.31, 95 % CI = 1.07–1.61, P = 0.010, P heterogeneity

Genetic epidemiological data in hepatocellular carcinoma (HCC) pedigrees indicate a pattern of X-linked recessive inheritance of HCC susceptibility genes. This study is designed to test the hypothesis that there are genes conferring susceptibility to HCC located on the X-chromosome.An X-chromosomal association study was conducted among Chinese men recruited from an area with a high prevalence of HCC. The candidate gene was further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).By analyzing 5454 X-chromosome single nucleotide polymorphisms (SNPs) in 50 HCC patients and 50 controls, we found two promising regions in which the associated SNPs clustered, located at Xq22.1 and Xq26.2. We further selected 35 tag SNPs (tSNPs) from these two regions for additional genotyping analysis in another independent set of 290 cases and 242 controls. Notably, SNP rs5945919 at Xq22.1 exhibited a significant association with HBV-related HCC (odds ratio [OR]=2.22, 95% confidence interval [CI]=1.15-4.30, P=0.016). The expressions of the three genes near the rs5945919 locus, RAB40AL, BEX1, and NXF3, were analyzed by qRT-PCR between another 24 HCC tissues and paired peritumoral liver tissues. The results indicated that NXF3, rather than RAB40AL and BEX1, mRNA level was found to be more abundant in HCC tissue than in peritumoral liver tissue.Our findings implicated Xq22.1 as a novel susceptibility locus for HCC and NXF3 as a candidate risk factor for relevant HCC.

Virus-induced hepatocarcinogenesis involves a series of histological developmental processes with the stepwise acquisition of several genetic changes that are necessary for the malignant transformation of hepatocytes. Although genetic alterations are known to be involved in the pathogenesis of hepatocellular carcinoma (HCC), little is known about the contributions of specific genes to this process. To gain insight into the genetic alterations involved in the neoplastic evolution from chronic hepatitis B virus infection to dysplastic nodules (DN) to HCC, we captured and sequenced the exomes of four DNA samples: one DN sample, two HCC samples, and one control peripheral blood sample from a single HCC patient. Mutations in the UBE3C gene (encoding ubiquitin ligase E3C) were observed in both tumor tissues. Then we resequenced the UBE3C gene in a cohort of 105 HCC patients and identified mutations in 17 out of a total of 106 (16.0%) HCC patients. The subsequent experiments showed that UBE3C promoted HCC progression by regulating HCC cells epithelial-mesenchymal transition. Clinically, a tissue microarray study of a cohort containing 323 HCC patients revealed that the overexpression of UBE3C in primary HCC tissues correlated with decreased survival (hazard ratio [HR] = 1.657, 95% confidence interval [CI] = 1.220-2.251, P = 0.001) and early tumor recurrence (HR = 1.653, 95% CI = 1.227-2.228, P = 0.001) in postoperative HCC patients. Conclusion: Our findings indicate that UBE3C is a candidate oncogene involved in tumor development and progression and therefore a potential therapeutic target in applicable HCC patients. (Hepatology 2014;59:2216–2227)

BACKGROUND: Sorafenib has become the standard first-line treatment for patients with advanced hepatocellular carcinoma (HCC). This study aimed to assess the efficacy and safety of sorafenib in advanced HCC patients and explore its true value for specific subgroups. DATA SOURCES: A computer-based systematic search from January 2005 to June 2011 with ”sorafenib” and ”advanced hepatocellular carcinoma” as search terms was performed for possible clinical trials. Hazard ratios (HR) and their 95% confidence intervals (CI) for overall survival (OS) and time to progression (TTP), rates of partial response (PR), rates of toxicity effects, and details of subgroup analysis were extracted. Meta-analyses were done using the software Review Manager (version 5.0). RESULTS: Six trials with 1164 patients were included. Based on three randomized controlled trials, the pooled HR (sorafenib/ placebo) was 0.66 for OS (95% CI: 0.56-0.78; P＜0.00001) and 0.57 for TTP (95% CI: 0.47-0.68; P＜0.00001). The pooled odds ratio (OR) for PR was 2.96 (95% CI: 0.96-9.15; P=0.06). For three single-arm trials, the pooled HR was 0.69 for OS (95% CI: 0.56-0.84; P=0.0002) and 0.64 for TTP (95% CI: 0.52-0.78; P＜0.00001). The pooled OR for PR in three single-arm trials was 3.56 (95% CI: 1.22-10.39; P=0.02). Subgroup analysis indicated that sorafenib was less effective in patients with extrahepatic spread (with: P=0.13 vs without: P＜0.0001), with normal alpha-fetoprotein level (AFP) (P=0.15 vs elevated: P=0.0006), and with elevated level of serum bilirubin (P=0.06 vs normal: P=0.0009). Sorafenib-based therapy significantly increased the risk of grade 3/4 hand-foot skin reaction, diarrhea, fatigue, and rash/desquamation. CONCLUSIONS: Sorafenib-based therapy benefits advanced HCC patients. Meanwhile, sorafenib is less effective for patients with extrahepatic spread, with normal AFP level and with elevated level of bilirubin.

Background In vitro experiments and mice models have confirmed the importance of Sprouty 2 (Spry2) in inhibiting tumorigenesis and the progression of human cancer. However, the prognostic value of Spry2 in cancer patients remains unknown. This study is aimed to investigate the clinical relevance and prognostic significance of Spry2 expression in patients with hepatocellular carcinoma (HCC). Methods With samples from 240 randomly-selected HCC patients who underwent surgery, immunohistochemistry was used to investigate Spry2 expression on tissue microarrays. The correlation of Spry2 expression with survival was estimated by the Kaplan-Meier method and univariate/multivariate Cox proportional hazard regression analysis. Spry2, ERK and phospho-ERK expression in HCC cell lines was detected by Western blotting. Results Among the patients, 86.3% (207 of 240) exhibited down-regulation of Spry2 expression. Patients negative for Spry2 showed poorer survival (P=0.002) and increased recurrence (P=0.003). Multivariate analysis further established Spry2 as an independent predictor of postoperative recurrence in HCC patients (HR=1.47; 95% CI, 1.02-2.08; P=0.037). Down-regulation of Spry2 was associated with highly malignant phenotypes like vascular invasion and advanced tumor stages, and was positively correlated with the metastatic potential of HCC cell lines. Conclusion In the era of molecular targeted therapy, the expression of Spry2 in HCC may have relevant clinical significance and turn out to be a key factor in prognostic assessment and in treatment planning.

Purpose More than 60% of patients with hepatocellular carcinoma (HCC) do not receive curative therapy as a result of late clinical presentation and diagnosis. We aimed to identify plasma microRNAs for diagnosing hepatitis B virus (HBV) –related HCC. Patients and Methods Plasma microRNA expression was investigated with three independent cohorts including 934 participants (healthy, chronic hepatitis B, cirrhosis, and HBV-related HCC), recruited between August 2008 and June 2010. First, we used microarray to screen 723 microRNAs in 137 plasma samples for diagnosing HCC. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using a training cohort (n = 407) and then validated using an independent cohort (n = 390). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. Results We identified a microRNA panel (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) that provided a high diagnostic accuracy of HCC (AUC = 0.864 and 0.888 for training and validation data set, respectively). The satisfactory diagnostic performance of the microRNA panel persisted regardless of disease status (AUCs for Barcelona Clinic Liver Cancer stages 0, A, B, and C were 0.888, 0.888, 0.901, and 0.881, respectively). The microRNA panel can also differentiate HCC from healthy (AUC = 0.941), chronic hepatitis B (AUC = 0.842), and cirrhosis (AUC = 0.884), respectively. Conclusion We found a plasma microRNA panel that has considerable clinical value in diagnosing early-stage HCC. Thus, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy.

The goal of this study is to investigate the effects of sorafenib on tumor growth, recurrence and metastasis after curative resection of liver cancer.SMMC-7721 and HCCLM3 liver tumors, each with different metastatic potential and basal phosphorylated extracellular signal-regulated kinase (pERK) levels, were orthotopically implanted into 56 nude mice. Mice were divided into a treatment sub study and a prevention sub study.In the treatment sub study, tumor volumes in the high pERK-expressing HCCLM3 model were 2.58 ? 0.83 and 0.38 ? 0.09 cm(3) without and with sorafenib, respectively (p0.001). The corresponding volumes in the low pERK-expressing SMMC-7721 model were 1.36 ? 0.24 and 0.24 ? 0.14 cm(3) (p0.001), respectively. Sorafenib inhibited HCCLM3 cell proliferation and decreased tumor angiogenesis, but did not inhibit proliferation in the SMMC-7721 model. In the prevention sub study, intrahepatic recurrent tumor volumes were 1.96 ? 0.45 and 0.18 ? 0.24 cm(3) (p0.001); lung metastasis frequencies were 100 and 0% (p = 0.005); and lifespans were 36 ? 3 and 46 ? 5 days (p = 0.002) in the control and sorafenib subgroups, respectively.Sorafenib inhibits tumor growth and prevents metastatic recurrence after resection of hepatocellular carcinoma in nude mice. The effect of sorafenib does not exclusively depend on high levels of pERK in tumors.

Proteomic techniques are promising strategies in the surveillance of hepatocellular carcinoma (HCC). This study aimed to investigate the serum profiling with magnetic bead (MB) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and to further identify the biomarkers for HCC. Serum samples from 80 chronic hepatitis B (CHB) patients, 94 HCC concomitant with HBV patients and 24 healthy subjects were examined by MALDI-TOF MS after peptide enrichment on MBs. Based on the genetic algorithm, diagnostic models for HCC were established between 30 HCC patients and 24 healthy subjects/30 CHB patients. Validations were done with the remaining cases. Markers in the models were identified through liquid chromatography (LC)/MS – MS. The three groups were well separated from each other and two discrimination models were established for HCC. The overall recognition capability of these two models was 96.25% and 93.33%, respectively. Validations showed the misdiagnosis ratio for HCC was 1.6% and 23.4%, respectively. The identified biomarkers for HCC included prothrombin precursor (fragment), calcium-dependent secretion activator 1, Baculoviral inhibitor of apoptosis repeat-containing protein 6, etc. MB-based MALDI-TOF MS is applicable in identifying the serum biomarkers and can be used in the surveillance of HCC among HBV-infected patients.

The density of tumor-infiltrating immunocytes (TICs) has been proposed as an independent predictor of intrahepatic recurrence in patients with hepatocellular carcinoma (HCC). However, the relative roles of TIC density in predicting tumor extrahepatic metastasis remain to be elucidated. The densities of CD3+, CD8+, granzyme B+, FoxP3+, CD45RO+, CD20+, CD1a+, CD83+, CD57+, and CD68+ TICs were assessed by immunohistochemistry in tissue microarrays containing paired intratumoral (IT) and peritumoral (PT) tissues from 206 consecutive HCC patients who underwent liver transplantation. Occurrence of extrahepatic metastasis, recurrence-free survival (RFS), and cancer-specific survival (CSS) were assessed retrospectively in relation to TIC densities. CD45RO+ memory T cell density was lower in tumor tissue compared with peritumor, whereas CD57+ senescent T cell density was higher. Univariate analysis revealed that increased CD45RO IT + and decreased CD57 PT + densities were statistically significantly associated with favorable RFS and CSS, while other types of TICs, intratumorally or peritumorally, showed no prognostic values. Further, the CD45RO IT + /CD57 PT + ratio could stratify patients more accurately in terms of RFS and CSS than either marker used alone. Finally, multivariate analysis indicated that a high CD45RO IT + /CD57 PT + ratio was independently associated with better RFS (hazard ratio [HR] = 0.64; 95% confidence interval [CI], 0.42 to 0.98; P = 0.040) and CSS (HR = 0.51; 95% CI, 0.31 to 0.83; P = 0.007), but not CD45RO IT + or CD57 PT + individually. These results suggest that the CD45RO IT + /CD57 PT + (memory/senescent T cell) ratio is of vital importance in preventing HCC extrahepatic metastasis and in particular demonstrates its independent prognostic value in liver transplant recipients.

To investigate the most suitable housekeeping genes for quantifying a change in mRNA expression levels due to hepatitis virus B related hepatocellular carcinoma (HCC). Expression of mRNA encoding ACTB, GAPDH, B2M, HPRT and TBP was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in matched malignant and non-malignant tissues obtained from 65 non-treated HCCs. The software programs geNorm and NormFinder were used to ascertain the most suitable reference gene combination. All candidate genes showed significantly different expression between malignant and non-malignant samples. GAPDH and ACTB, genes most frequently used for normalization, were heavily regulated during HCC carcinogenesis and progression. B2M expression levels varied with hepatitis infection status. The combination of HPRT and TBP expression levels were the most stable, regardless of differences in tumor stage and cirrhotic and malignancy status. It is necessary to select reference genes based on tissue and disease specific expression profile and to further identify novel reference genes with greater expression stability for use in HBV related HCC gene expression studies.

Background Recurrence prediction of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) present a great challenge because of a lack of biomarkers. Genetic variations play an important role in tumor development and metastasis. Methods Oligonucleotide microarrays were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent LT. Recurrence-related single-nucleotide polymorphism were selected, and their prognostic value was assessed and validated in two independent cohorts of HCC patients (N = 102 and N = 77), using pretransplant plasma circulating DNA. Prognostic significance was assessed by Kaplan-Meier survival estimates and log-rank tests. Multivariate analyses were performed to evaluate prognosis-related factors. Results rs894151 and rs12438080 were significantly associated with recurrence (P = .003 and P = .004, respectively). Multivariate analyses demonstrated that the co-index of the 2 SNPs was an independent prognostic factor for recurrence (P = .040). Similar results were obtained in the third cohort (N = 77). Furthermore, for HCC patients (all the 3 cohorts) exceeding Milan criteria, the co-index was a prognostic factor for recurrence and survival (P

AIM: To report a retrospective analysis of preliminary results of 36 patients who received sirolimus (SRL, Rapamune®, rapamycin) in a consecutive cohort of 248 liver allograft recipients. METHODS: Thirty-six liver transplant patients with hepatocellular carcinoma (HCC) who were switched to SRL-based immunosuppression therapy from tacrolimus were enrolled in this study. The patients who were diagnosed as advanced HCC before orthotopic liver transplantation (OLT) were divided into group A (n = 11), those who were found to have HCC recurrence and/or metastasis after OLT were assigned to group B (n = 18), and those who developed renal insufficiency caused by calcineurin inhibitor (CNI) were assigned to group C (n = 7) after OLT. RESULTS: The patients were followed up for a median of 10.4 mo (range, 3.8-19.1 mo) after conversion to SRL therapy and 12.3 mo (range, 5.1-34.4 mo) after OLT. Three patients developed mild acute cellular rejection 2 wk after initiating SRL therapy, which was fully reversed after prednisolone pulse therapy. In group A, only 1 patient was found to have HCC recurrence and metastasis 12 mo after OLT. In group B, 66.7% (12/18) patients (2 with progressive tumor, 7 with stable tumor and 3 without tumor) were still alive due to conversing to SRL and/or resection for HCC recurrence at the end of a median follow-up of 6.8 mo post conversion and 10.7 mo posttransplant. In group C, no HCC recurrence was demonstrated in 7 patients, and renal function became normal after SRL therapy. Thrombocytopenia (n = 2), anemia (n = 8), and oral aphthous ulcers (n = 7) found in our cohort were easily manageable. CONCLUSION: The conversion to SRL-based immunosuppression may inhibit the recurrence and metastasis of HCC and improve CNI-induced renal insufficiency in OLT patients with HCC.

Hepatocellular carcinoma(HCC) is a significant health problem in China. But the molecular mechanisms of HCC remains unclear. APC/beta-Catenin/Tcf signaling pathway, also known as Wnt pathway, plays a critical role in the development and oncogenesis. As little is known about the alteration of human T-cell transcription factor-4 (hTcf-4) gene in HCC, it is of interest to study the expression and mutation of hTcf-4 gene in HCC and the relationship between hTcf-4 gene and progression of HCC.Reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of hTcf-4 mRNA in 32 HCC and para-cancerous tissues and 5 normal liver tissues. PCR-single strand conformation polymorphism (PCR-SSCP) method was used to detect the mutation of hTcf-4 exons 1, 4, 9 and 15 in HCC. The correlation of expression and mutation of the hTcf-4 gene with clinicopathological characteristics of HCC was also analyzed.RT-PCR showed that the expression rate of hTcf-4 mRNA in HCC, para-cancerous tissues and normal liver tissues was 90.6 %, 71.9 % and 80 %, respectively. The gene expression level in tumor was 0.71+/-0.13, much higher than that in para-cancerous liver 0.29+/-0.05 and normal liver 0.26+/-0.05 (P0.001), although there was no significant difference in gene expression level between para-cancerous tissues and normal liver (P0.05). Furthermore, hTcf-4 gene expression was closely associated with tumor capsule status and intrahepatic metastasis of HCC. On SSCP, 2 of 32 cases of HCC (6.25 %) displayed characteristic mutational mobility shifts in exon 15 of the hTcf-4 gene. No abnormal shifting bands were observed in para-cancerous tissues.The high expression level of hTcf-4 in HCC, especially in tumors with metastasis, suggests that the over-expression of hTcf-4 gene may be closely associated with development and progression of HCC, but the mutation of this gene seemed to play less important role in this respect.