Variants Identified by Hepatocellular Carcinoma and Chronic Hepatitis B Virus Infection Susceptibility GWAS Associated with Survival in HBV-Related Hepatocellular Carcinoma

Recent genome-wide association studies (GWAS) have identified several common susceptibility loci associated with the risk of hepatocellular carcinoma (HCC) or chronic hepatitis B infection (CHB). However, the relationship between these genetic variants and survival of patients with hepatitis B virus (HBV)-related HCC is still unknown. In this study, 22 single nucleotide polymorphisms (SNPs) were genotyped among 330 HBV-related HCC patients using the MassARRAY system from Sequenom. Cox proportional hazards regression was used to examine the effects of genotype on survival time under an additive model with age, sex, smoking status and clinical stage as covariates. We identified four SNPs on 6p21 (rs1419881 T>C, rs7453920 G>A,rs3997872 G>A and rs7768538 T>C), and two SNPs on 8p12 (rs2275959 C>T and rs7821974 C>T) significantly associated with survival time of HBV-related HCC patients. Our results suggest that HCC or CHB susceptibility loci might also affect the prognosis of patients with HBV-related HCC.     

Batimastat Nanoparticles Associated with Transcatheter Arterial Chemoembolization Decrease Hepatocellular Carcinoma Recurrence

Hepatocellular carcinoma (HCC) is a malignant tumor characterized by easy metastasis and frequent recurrence. Transarterial chemoembolization (TACE) remains the routine treatment for patients with HCC who are not eligible for surgical resection or percutaneous tumor ablation; however, 5-year survival rates following interventional therapy are only 17–38.8 %, with liver recurrence due to incomplete embolization and tumor angiogenesis being a significant reason for treatment failure. Ischemia and hypoxia induced by TACE is correlated with an increased expression of angiogenic factor and stimulates an increase in angiogenesis, including endothelial cells (ECs) proliferation. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic endopeptidases involved in tumor angiogenesis. In addition, MMPs stimulate tumor cell growth, migration and invasion, and metastasis. Hypoxia enhanced EC migration in a MMP-2-dependent manner while MMP inhibitors (MMPIs) significantly decreased the number of migrating cells in hypoxic cultures. We hypothesize batimastat (synthetic MMPI) nanoparticles associated with TACE could decrease HCC recurrence and metastasis. At first, batimastat nanoparticles were made from batimastat and poly(lactic-co-glycolic acid). Then, nanoparticles were mixed with lipiodol and chemotherapeutic drugs solution. The mixture was infused super-selectively into supplied artery of HCC through catheter. The disseminated area of batimastat might be same with TACE-induced hypoxia area. In the hypoxia area, batimastat inhibited the activity of MMPs, weakened the angiogenesis of tumor vascular system and migration of HCC cells. HCC cells could not escape from hypoxia area and tumor angiogenesis inhibited could not supply sufficient nutrients and O₂ to residual HCC cells. With the help of batimastat, the killing effect of chemotherapeutic drugs might be enhanced. The rate of complete necrosis of HCC lesion might be increased and local recurrence and metastasis of HCC might be reduced. The hypothesis might increase the clinical efficacy of TACE and improve the prognosis of HCC patients.     

Embryonic Morphogen Nodal Is Associated with Progression and Poor Prognosis of Hepatocellular Carcinoma

Background

Nodal, a TGF-β-related embryonic morphogen, is involved in multiple biologic processes. However, the expression of Nodal in hepatocellular carcinoma (HCC) and its correlation with tumor angiogenesis, epithelial-mesenchymal transition, and prognosis is unclear.

Methods

We used real-time PCR and Western blotting to investigate Nodal expression in 6 HCC cell lines and 1 normal liver cell line, 16 pairs of tumor and corresponding paracarcinomatous tissues from HCC patients. Immunohistochemistry was performed to examine Nodal expression in HCC and corresponding paracarcinomatous tissues from 96 patients. CD34 and Vimentin were only examined in HCC tissues of patients mentioned above. Nodal gene was silenced by shRNA in MHCC97H and HCCLM3 cell lines, and cell migration and invasion were detected. Statistical analyses were applied to evaluate the prognostic value and associations of Nodal expression with clinical parameters.

Results

Nodal expression was detected in HCC cell lines with high metastatic potential alone. Nodal expression is up-regulated in HCC tissues compared with paracarcinomatous and normal liver tissues. Nodal protein was expressed in 70 of the 96 (72.9%) HCC tumors, and was associated with vascular invasion (P = 0.000), status of metastasis (P = 0.004), AFP (P = 0.049), ICGR₁₅ (indocyanine green retention rate at 15 min) (P = 0.010) and tumor size (P = 0.000). High Nodal expression was positively correlated with high MVD (microvessal density) (P = 0.006), but not with Vimentin expression (P = 0.053). Significantly fewer migrated and invaded cells were seen in shRNA group compared with blank group and negative control group (P<0.05). High Nodal expression was found to be an independent factor for predicting overall survival of HCC.

Conclusions

Our study demonstrated that Nodal expression is associated with aggressive characteristics of HCC. Its aberrant expression may be a predictive factor of unfavorable prognosis for HCC after surgery.     

Positive Lymph Node Metastasis Has a Marked Impact on the Long-Term Survival of Patients with Hepatocellular Carcinoma with Extrahepatic Metastasis

Background

The prognosis of hepatocellular carcinoma (HCC) patients with extrahepatic metastasis is extremely poor. However, what is the main risk factor for survival remains unclear for these patients. We aimed to find out the relative frequency, incidence and locations of extrahepatic metastases and the risk factors of long-term survival of the patients.

Methods

132 HCC patients with extrahepatic metastasis diagnosed by ¹⁸F-FDG PET/CT and conventional workup were enrolled into this study. The incidence and locations of extrahepatic metastases were summarized, and the related risk factors of overall survival were analyzed.

Results

The most frequent extrahepatic metastatic sites were lymph nodes in 72 (54.5%), bone in 33 (25.0%) and lung in 28 (21.2%) patients. On univariate analysis, prothrombin time, Child-Pugh grade, portal/hepatic vein invasion and lymph node metastasis were independent risk factors of overall survival. On multivariate analysis, lymph node metastasis was the only independent risk factor of overall survival. The cumulative survival rates at 1- and 3-years after diagnosis of extrahepatic metastasis of HCC were 34.4% and 9.3%, respectively. The median survival time was 7 months (range 1 ∼38 months). The median survival time for patients with or without lymph node metastasis were 5 months (range 1∼38 months) and 12 months (range 1∼30 months), respectively (P = 0.036).

Conclusions

This study showed lymph nodes to be the most frequent site of extrahepatic metastases for primary HCC. Lymph node metastasis was the main risk factor of overall survival in patients with HCC with extrahepatic metastasis.     

肝细胞肝癌E-Cadherin蛋白的表达及其对判断肝癌肝移植疗效的意义

目的：探讨E-Cadherin蛋白在肝细胞肝癌组织中的表达及其对判断肝癌肝移植患者预后的价值。方法：选择肝细胞肝癌行全肝移植患者68例,应用免疫组化方法检测其切除的肝癌组织和癌旁正常肝组织中E-Cadherin蛋白的表达情况,并对患者进行移植术后随访,分析E-Cadherin蛋白表达与肝癌肝移植患者预后的关系。结果：肝癌组织中E-Cadherin蛋白阳性表达率明显低于癌旁组织（P〈0.01）。经Logrank检验分析显示,E-Cadherin蛋白低表达组移植后的无瘤生存率明显低于E-Cadherin蛋白高表达组（P〈0.01）。多因素Cox回归分析显示,E-Cadherin蛋白表达是影响肝细胞肝癌患者肝移植术后肝癌复发的独立预后因素之一。结论：E-Cadherin蛋白表达是一个预测肝细胞肝癌患者肝移植预后的重要因子。     

Elastin Fiber Accumulation in Liver Correlates with the Development of Hepatocellular Carcinoma

MethodsWe enrolled 189 consecutive patients with hepatitis C and advanced fibrosis. Using Elastica van Gieson-stained whole-slide images of pretreatment liver biopsies, collagen and elastin fibers were evaluated pixel by pixel (0.46 μm/pixel) using an automated computational method. Consequently, fiber amount and cumulative incidences of HCC within 3 years were analyzed.ResultsThere was a significant correlation between collagen and elastin fibers, whereas variation in elastin fiber was greater than in collagen fiber. Both collagen fiber (p = 0.008) and elastin fiber (p < 0.001) were significantly correlated with F stage. In total, 30 patients developed HCC during follow-up. Patients who have higher elastin fiber (p = 0.002) in addition to higher collagen fiber (p = 0.05) showed significantly higher incidences of HCC. With regard to elastin fiber, this difference remained significant in F3 patients. Furthermore, for patients with a higher collagen fiber amount, higher elastin was a significant predictor for HCC development (p = 0.02).ConclusionsComputational analysis is a novel technique for quantification of fibers with the added value of conventional staging. Elastin fiber is a predictor for the development of HCC independently of collagen fiber and F stage.     

Comparison of Steroid-Free Immunosuppression and Standard Immunosuppression for Liver Transplant Patients with Hepatocellular Carcinoma

Immunosuppression therapy following liver transplantation often includes steroids. However, extended corticosteroid therapy is associated with numerous complications. This study evaluated the efficacy and safety of using basiliximab in place of a corticosteroid for immunosuppression following liver transplantation for hepatocellular carcinoma (HCC) in Chinese patients. The records of 178 patients with HCC who underwent orthotopic liver transplantation from January 2003 to December 2009 were retrospectively reviewed. All patients received immunosuppression therapy that contained either basiliximab (n = 78) or steroids (n = 100) in addition to tacrolimus and mycophenolate mofetil. Assessments included complications related to liver transplantation, occurrence of steroid side effects, recurrence of HCC, and patient and graft survival. A smaller proportion of patients receiving basiliximab compared with steroids experienced de novo diabetes (38.7% vs. 91.0%, respectively) or long-term de novo diabetes mellitus (7.7% vs. 38.0%, respectively) (both, P<0.0001). The median overall and disease free survival was similar between basiliximab (50.8 months and 19.6 months, respectively) and steroid treated patients (64.2 months and 23.8 months, respectively). The 5-year overall survival and disease free survival rates was also similar between the basiliximab (42.5% and 38.9%, respectively) and steroid (50.5% and 39.2%) groups (all, P>0.730). However, in patients who met the Milan criteria basiliximab was associated with greater 5-year overall survival rate as compared with steroid therapy (88.9% vs. 57.4%, respectively, P = 0.022). These findings provide further evidence of the negative impact of steroids as a part of immunosuppression therapy following liver transplantation for HCC.     

FBX8 Acts as an Invasion and Metastasis Suppressor and Correlates with Poor Survival in Hepatocellular Carcinoma

Background

F-box only protein 8 (FBX8), a novel component of F-box proteins, is lost in several cancers and has been associated with invasiveness of cancer cells. However, its expression pattern and role in the progression of hepatocellular carcinoma remain unclear. This study investigated the prognostic significance of FBX8 in hepatocellular carcinoma samples and analyzed FBX8 function in hepatocellular carcinoma cells by gene manipulation.

Methodology

The expression of FBX8 was detected in 120 cases of clinical paraffin-embedded hepatocellular carcinoma tissues, 20 matched pairs of fresh tissues and five hepatocellular carcinoma cell lines by immunohistochemistry with clinicopathological analyses, real-time RT-PCR or Western blot. The correlation of FBX8 expression with cell proliferation and invasion in five HCC cell lines was analyzed. Moreover, loss of function and gain of function assays were performed to evaluate the effect of FBX8 on cell proliferation, motility, invasion in vitro and metastasis in vivo.

Conclusions

We found that FBX8 was obviously down-regulated in HCC tissues and cell lines (P<0.05). The FBX8 down-regulation correlated significantly with poor prognosis, and FBX8 status was identified as an independent significant prognostic factor. Over-expression of FBX8 decreased proliferation, migration and invasion in HepG2 and 97H cells, while knock-down of FBX8 in 7721 cells showed the opposite effect. FBX8 negatively correlated with cell proliferation and invasion in 7701, M3, HepG2 and 97H cell lines. In vivo functional assays showed FBX8 suppressed tumor growth and pulmonary metastatic potential in mice. Our results indicate that down-regulation of FBX8 significantly correlates with invasion, metastasis and poor survival in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy and control in hepatocellular carcinoma treatment.     

脾切除术对肝炎后肝硬化患者肝癌发生的影响

目的:观察脾切断流术对肝硬化门静脉高压症患者肝功的影响.方法:回顾分析2010.4-2011.12在我科住院的肝硬化门静脉高压症患者行脾切除术前后肝功能的变化.应用超声多普勒分别检测手术前后门静脉血流量(PVF)、肝动脉血流量(HAF),术中水柱法测量自由门静脉压力(FPP);回顾分析(通过对病历查阅获取)上述患者Child分级指标以及肝功能指标.用SAS 8.0对获取的指标采用配对资料t检验和卡方检验.结果:肝硬化门静脉高压症患者共480例,行脾脏切除加贲门周围血管离断术450例,单纯脾切除30例.PVF由术前的(1368± 402)ml/min减少至术后的(986± 295)ml/min(P＜0.01);HAF由术前的(412± 68)ml/min增加至术后的(526± 132)ml/min(P＜0.01);FPP由术前的(30± 6)cm H2O降至术后的(26± 5)cm H2O(P＜0.01).R15 ICG由术前的(18±8)％降至术后的(16±7)％(P＞0.05).肝功能Child分级A级患者比率由术前85％增加至术后95％(P＜0.01).其它肝脏功能指标如总胆红素、凝血酶原延长时间以及腹水消失率都明显好转.结论:脾切断流术后门静脉血流量虽然减少,但是肝动脉血流量增加及术后门体分流减少均有利于肝功能恢复.     

Positive Expression of Programmed Death Ligand 1 in Peritumoral Liver Tissue is Associated with Poor Survival after Curative Resection of Hepatocellular Carcinoma

BACKGROUND: Recurrence or metastasis of hepatocellular carcinoma (HCC) is mainly intrahepatic after curative resection, demonstrating that the peritumoral environment is important but often neglected. Programmed death ligand 1 (PD-L1) in intratumoral liver tissues is a poor prognosis factor whose impact is removed after curative resection. However, PD-L1 expression remains in the peritumoral liver tissues and its distribution and prognostic value are still not clear. METHODS: We assessed the expression of PD-L1 by immunohistochemistry in peritumoral liver tissues from 90 HCC patients who underwent curative hepatectomy. The results were validated in an independent cohort of additional 90 HCC patients. RESULTS: We found PD-L1 positive expression in 31.11% (28/90) of peritumoral tissues. Peritumoral PD-L1 expression was associated with a significantly worse overall survival (OS) (P = .000) and disease-free survival (DFS) (P = .001) compared to the negative expression group. Additionally, peritumoral PD-L1 positivity significantly correlated with vascular invasion and a lower albumin level (≤35 g/L). Univariate and multivariate Cox regression models both revealed peritumoral PD-L1 as an independent prognostic factor for OS (HR = 2.853, P = .002) and DFS (HR = 2.362, P = .003). The prognostic value of PD-L1 positivity was validated in the independent data set. CONCLUSIONS: Our data suggest PD-L1 expression in peritumoral hepatocytes is an independent prognostic factor for OS and DFS. This implies that future anti-cancer therapy should target not only residual tumor cells but also the “soil” for promoting tumor growth. Peritumoral PD-L1 could be a good target for adjuvant therapy after hepatectomy.     

Gamma-Smooth Muscle Actin Expression Is Associated with Epithelial-Mesenchymal Transition and Stem-Like Properties in Hepatocellular Carcinoma

Background and Aims

The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.

Methods

The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.

Results

We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.

Conclusion

Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.     

Prediction of Post-Operative Liver Dysfunction by Serum Markers of Liver Fibrosis in Hepatocellular Carcinoma

AimTo investigate the role of biomarkers in predicting postoperative liver dysfunction in patients with hepatocellular carcinoma (HCC).MethodsA total of 200 patients operated from July 2009 to June 2010 at Zhongshan Hospital, Fudan University for pathologically confirmed HCC were retrospectively analyzed for clinical data, HBD DNA level and serum biochemical markers for liver fibrosis. The patients were followed up to observersation end point. Correlation of the monitored parameters with postoperative liver dysfunction and patient survival was statistically analyzed.ResultsPreoperative hepatitis B virus (HBV) DNA level, serum prealbumin (PA) hyaluronic acid (HA), and laminin (LN) levels correlated with postoperative liver dysfunction. A predictive model was generated using these 4 parameters and validated in 89 HCC patients with sensitivity and specificity of 0.625 and 0.912, respectively. However, no correlation was identified between postoperative liver function and overall survival.ConclusionLiver fibrosis markers could be preoperatively used in predicting postoperative liver dysfunction in HCC patients.     

Association of Serum Level of Growth Differentiation Factor 15 with Liver Cirrhosis and Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) and liver cirrhosis are associated with high mortality worldwide. Currently, alpha-fetoprotein (AFP) is used as a standard serum marker for the detection of HCC, but its sensitivity and specificity are unsatisfactory, and optimal diagnostic markers for cirrhosis are lacking. We previously reported that growth differentiation factor 15 (GDF15) was significantly induced in HCV-infected hepatocytes. This study aimed to investigate GDF15 expression and its correlation with hepatitis virus-related liver diseases. A total of 412 patients with various liver diseases were studied. Healthy and Mycobacterium tuberculosis-infected subjects were included as controls. Serum and tissue GDF15 levels were measured. Serum GDF15 levels were significantly increased in patients with HCC (6.66±0.67 ng/mL, p<0.0001) and cirrhosis (6.51±1.47 ng/mL, p<0.0001) compared with healthy controls (0.31±0.01 ng/mL), though the GDF15 levels in HBV and HCV carriers were moderately elevated (1.34±0.19 ng/mL and 2.13±0.53 ng/mL, respectively). Compared with HBV or HCV carriers, GDF15 had a sensitivity of 63.1% and a specificity of 86.6% at the optimal cut-off point of 2.463 ng/mL in patients with liver cirrhosis or HCC. In HCC patients, the area under the receiver operating curve was 0.84 for GDF15 and 0.76 for AFP, but 0.91 for the combined GDF15 and AFP. Serum GDF15 levels did not significantly differ between the high-AFP and low-AFP groups. GDF15 protein expression in HCC was significantly higher than that in the corresponding adjacent paracarcinomatous tissue and normal liver. Using a combination of GDF15 and AFP will improve the sensitivity and specificity of HCC diagnosis. Further research and the clinical implementation of serum GDF15 measurement as a biomarker for HCC and cirrhosis are recommended.