Abnormal Expression of Golgi Protein 73 in Clinical Values and Their Role in HBV-Related Hepatocellular Carcinoma Diagnosis and Prognosis

Background:

The up-regulation of hepatic Golgi protein 73 (GP73) is associated with the progression of hepatocellular carcinoma (HCC). However, the exact mechanism and clinical values of its diagnosis and prognosis still need to be clarified.

Objectives:

To investigate the clinical values of abnormal liver or circulating GP73 expression and their effect on HCC diagnosis and prognosis.

Materials and Methods:

The expression of GP73 was investigated in 88 cancerous and self-control non-cancerous tissues using tissue microarrays with immunohisto- chemistry and was confirmed by Western blotting. Circulating GP73 levels were detected in the sera of 281 patients with liver diseases using enzyme-linked immunosorbent assay.

Results:

The levels of circulating GP73 expression in the HCC group were higher than those in any group of benign liver diseases or controls. No significant difference was found between GP73 expression and patients’ sex or age, tumor size, or AFP level except for those with hepatitis B virus (HBV) infection or distal metastasis (P < 0.05). The area under the receiver operating characteristic curve, sensitivity, and specificity for HCC diagnosis were 0.881, 78.34%, and 77.59% for GP73 levels over 70 μg/L or 0.754, 71.97%, and 84.48% for alpha-fetoprotein levels over 50 μg/L, respectively. The total incidence of GP73 plus alpha-fetoprotein was up to 87.26% for HCC. A positive GP73 result with brown particles was mainly located in the cytosol, with a few in the nucleus and none in the cell membrane, with abnormal expression in HCC tissues (480.7 ± 148.7) that was significantly higher (t = 10.730, P < 0.001) than those in their non-cancerous tissues (208.0 ± 66.1). The high GP73 expression in HCC was related to lymph node metastasis (χ² = 6.940, P = 0.008), gross classification (χ² = 6.311, P = 0.012), HBV (χ² = 4.803, P = 0.028), tumor node metastasis staging (χ² = 4.887, P = 0.027), and five-year survival (χ² = 5.206, P = 0.023).

Conclusions:

Abnormality of hepatic or circulating GP73 expression should be regarded as an emerging biomarker for HCC diagnosis and prognosis.     

Serum Golgi Protein 73 (GP73) is a Diagnostic and Prognostic Marker of Chronic HBV Liver Disease

Alanine aminotransferase (ALT) is the most commonly used marker of liver injury, but normal ALT levels are seen in a proportion of chronic hepatitis B virus (HBV)-infected patients with severe liver injury. Golgi protein 73 (GP73) is a promising alternative marker of liver injury. This study assessed the relation between GP73 levels and liver disease severity, monitored the kinetic changes in GP73 levels in chronic HBV patients receiving entecavir (ETV) therapy, and investigated the potential diagnostic and prognostic values of serum GP73 as a new liver injury biomarker in chronic HBV infections.This study enrolled 1150 patients with chronic HBV infections, 200 of whom were retrospectively enrolled in this study after receiving 1 year of ETV treatment. GP73 expression in liver tissue was detected by immunohistochemistry. GP73 levels in single or serial serum samples were measured by enzyme-linked immunosorbent assay.Immunohistochemical analysis indicated that GP73 protein expression in the liver increased progressively with pathologic progression from nonexistent or mild hepatitis to severe hepatitis and cirrhosis during chronic HBV infection. Serum GP73 levels were positively correlated with the disease severity of chronic HBV infections (r = 0.58, P < 0.001). In patients with normal ALT levels, serum GP73 concentrations were significantly higher in patients with prominent hepatic inflammatory injury and fibrosis than in patients without hepatic inflammatory injury or fibrosis. Serum GP73 concentrations and GP73 protein expression were decreased in the liver tissues of patients whose ALT levels normalized after 1 year of ETV antiviral therapy.Changes in serum GP73 levels were closely associated with changes in liver injury severity, and, therefore, GP73 may be an effective new liver inflammatory injury biomarker, and could be useful for monitoring the prognosis of chronic HBV infectious patients with normal ALT levels.     

Disease- and cell-specific expression of GP73 in human liver disease

OBJECTIVES: GP73, a Golgi membrane protein, is expressed at high levels in hepatocytes of patients with decompensated cirrhosis. Its expression in other forms of liver disease has not been investigated. Therefore, we studied GP73 expression in patients with noncirrhotic liver disease. METHODS: GP73 expression was detected immunohistochemically and by immunofluorescence microscopy in patients with acute hepatitis of various etiologies, autoimmune hepatitis, chronic HCV infection, and alcoholic liver disease. In order to quantitate hepatocyte GP73 expression, an immunohistochemical scoring system was developed, and validated by a direct comparison with GP73 protein levels as determined by Western blotting. RESULTS: GP73 immunostaining and Western blotting data were highly correlated, demonstrating the suitability of the immunohistochemical scoring system to quantitate hepatocyte GP73 expression. Hepatocyte GP73 expression was increased in patients with acute and autoimmune hepatitis. Treatment of autoimmune hepatitis was associated with a normalization of GP73 expression, indicating that the initial upregulation was reversible. Increased levels of GP73 expression were also noted in chronic HCV infection and alcoholic liver disease. Under these conditions, GP73 levels were correlated with disease stage but not grade. GP73 immunoreactivity was occasionally detected in alpha-SMA-positive, sinusoidal lining cells, suggesting activated stellate cells as a potential source of GP73. CONCLUSIONS: Hepatocyte GP73 levels are upregulated in acute hepatitis and during the progression of liver disease to cirrhosis. This expression pattern suggests the presence of two regulatory mechanisms, the first triggered during acute hepatocellular injury, the second during the progression of chronic liver disease.     

Overexpression of GP73 promotes cell invasion,migration and metastasis by inducing epithelial-mesenchymal transition in pancreatic cancer

Pancreatic cancer is one of the most difficult clinical cases to diagnose with a very low 5-year survival rate of 5%, regardless of the advances made in both the medical and surgical treatment of the disease. One of the contributing factors for the high mortality rate seen of pancreatic cancer patients is the lack of effective chemotherapies, which is believed to be due to drug-resistance. Based on recent evidence, epithelial-mesenchymal transition (ETM) of pancreatic cancer cells has been found to be associated with the development of drug resistance and an increase in cell invasion. Therefore, we conducted the present study in order to investigate the regulatory effects of Golgi protein-73 (GP73) on PC. GP73 and EMT-related gene expressions in PC, along with the adjacent and chronic pancreatitis tissues were determined by means of RT-qPCR and Western blot analysis. Cultured PC cells were treated with pAdTrack-CMV, si-NC, GP73 overexpression, Si-GP73, Snail-siRNA and GP73 + Snail-siRNA. Cell invasion, migration and metastasis were measured in vitro and in vivo. The results revealed that the PC tissues and chronic pancreatitis tissues exhibited diminished E-cadherin expression and amplified GP73, N-cadherin, Vimentin and Snail expression. In response to GP73 gene silencing, PC cells presented with increased E-cadherin expression and decreased N-cadherin, Vimentin, Snail expression in addition to the inhibition of the number of invasive cells, tumor volume and number of liver lesions. These findings highly indicated that the overexpression of GP73 promotes cell invasion, migration and metastasis by inducing EMT in PC.     

血清甲胎蛋白和高尔基体糖蛋白73联合检测对肝细胞癌的早期诊断价值

目的探讨血清甲胎蛋白(AFP)和高尔基体糖蛋白73(GP73)联合检测对肝细胞癌(HCC)的诊断价值,为早期诊断和鉴别诊断HCC提供依据。方法收集2012年6月-2013年5月住院患者标本共408例,其中HCC患者142例(HCC组),慢性肝炎患者156例(慢性肝炎组),肝硬化患者110例(肝硬化组)。分别采用电化学发光法和酶联免疫吸附试验(ELISA)双抗体夹心法测定3组患者的血清AFP和GP73的浓度,检测结果组间比较采用方差分析,率的比较采用χ2检验。并使用MedCalc统计学软件计算2项指标联合检测对HCC诊断的敏感度和特异度。结果 HCC组血清AFP和GP73的浓度显著高于肝硬化组和慢性肝炎组,差异具有统计学意义(P0.05);肝硬化组血清AFP和GP73的浓度显著高于慢性肝炎组,其差异亦有统计学意义(P0.05)。二者联合检测肝细胞癌的敏感度为95.8%,特异度为98.6%,较单项检测差异具有统计学意义(P0.05)。结论血清AFP和GP73联合检测对HCC具有较高的诊断价值和临床意义,可作为HCC早期的诊断和鉴别诊断指标,值得在临床上推广。     

Golgi protein-73: A biomarker for assessing cirrhosis and prognosis of liver disease patients

BACKGROUND Reliable biomarkers of cirrhosis, hepatocellular carcinoma(HCC), or progression of chronic liver diseases are missing. In this context, Golgi protein-73(GP73) also called Golgi phosphoprotein-2, was originally defined as a resident Golgi type II transmembrane protein expressed in epithelial cells. As a result, GP73 expression was found primarily in biliary epithelial cells, with only slight detection in hepatocytes. However, in patients with acute or chronic liver diseases and especially in HCC, the expression of GP73 is significantly up-regulated in hepatocytes. So far, few studies have assessed GP73 as a diagnostic or prognostic marker of liver fibrosis and disease progression.AIM To assess serum GP73 efficacy as a diagnostic marker of cirrhosis and/or HCC or as predictor of liver disease progression.METHODS GP73 serum levels were retrospectively determined by a novel GP73 ELISA (QUANTA Lite~? GP73, Inova Diagnostics, Inc., Research Use Only) in a large cohort of 632 consecutive patients with chronic viral and non-viral liver diseases collected from two tertiary Academic centers in Larissa, Greece(n = 366) and Debrecen, Hungary(n = 266). Aspartate aminotransferase(AST)/Platelets(PLT) ratio index(APRI) was also calculated at the relevant time points in all patients. Two hundred and three patients had chronic hepatitis B, 183 chronic hepatitis C, 198 alcoholic liver disease, 28 autoimmune cholestatic liver diseases, 15 autoimmune hepatitis, and 5 with other liver-related disorders. The duration of follow-up was 50(57) mo [median(interquartile range)]. The development of cirrhosis, liver decompensation and/or HCC during follow-up were assessed according to internationally accepted guidelines. In particular, the surveillance for the development of HCC was performed regularly with ultrasound imaging and alpha-fetoprotein(AFP) determination every 6 mo in cirrhotic and every 12 mo in non-cirrhotic patients.RESULTS Increased serum levels of GP73( 20 units) were detected at initial evaluation in 277 out of 632 patients(43.8%). GP73-seropositivity correlated at baseline with the presence of cirrhosis(96.4% vs 51.5%, P 0.001), decompensation of cirrhosis(60.3% vs 35.5%, P 0.001), presence of HCC(18.4% vs 7.9%, P 0.001) and advanced HCC stage(52.9% vs 14.8%, P = 0.002). GP73 had higher diagnostic accuracy for the presence of cirrhosis compared to APRI score [Area under the curve(AUC)(95%CI): 0.909(0.885-0.934) vs 0.849(0.813-0.886), P = 0.003]. Combination of GP73 with APRI improved further the accuracy(AUC: 0.925) compared to GP73(AUC: 0.909, P = 0.005) or APRI alone(AUC: 0.849, P 0.001). GP73 levels were significantly higher in HCC patients compared to non-HCC [22.5(29.2) vs 16(20.3) units, P 0.001) and positively associated with BCLC stage [stage 0: 13.9(10.8); stage A: 17.1(16.8); stage B: 19.6(22.3); stage C: 32.2(30.8); stage D: 45.3(86.6) units, P 0.001] and tumor dimensions [very early: 13.9(10.8); intermediate: 19.6(18.4); advanced: 29.1(33.6) units, P = 0.004]. However, the discriminative ability for HCC diagnosis was relatively low [AUC(95%CI): 0.623(0.570-0.675)]. Kaplan-Meier analysis showed that the detection of GP73 in patients with compensated cirrhosis at baseline, was prognostic of higher rates of decompensation(P = 0.036), HCC development(P = 0.08), and liver-related deaths(P 0.001) during follow-up.CONCLUSION GP73 alone appears efficient for detecting cirrhosis and superior to APRI determination. In combination with APRI, its diagnostic performance can be further improved. Most importantly, the simple GP73 measurement proved promising for predicting a worse outcome of patients with both viral and nonviral chronic liver diseases.     

GP73, a resident Golgi glycoprotein, is a novel serum marker for hepatocellular carcinoma

BACKGROUND/AIMS: Golgi protein-73 (GP73) is up-regulated in hepatocellular carcinoma (HCC). The aims of this study were to determine if GP73 is detected in the serum, and to establish the sensitivity and specificity of serum GP73 for diagnosing HCC. METHODS: Serum GP73 was detected by immunoblots and quantified by densitometric analysis. RESULTS: A total of 352 patients were studied. Serum GP73 levels were significantly higher in patients with HCC compared to those with cirrhosis (P < 0.001). GP73 had a sensitivity of 69% and a specificity of 75% at the optimal cutoff point of 10 relative units, with an area under the receiver operating curve of 0.79 vs. 0.61 for AFP (P = 0.001). GP73 levels had significantly higher sensitivity (62%) than AFP (25%) for diagnosing early HCC (P < 0.0001). Moreover, GP73 levels were elevated in the serum of 57% (32/56) of individuals with HCC who had serum AFP levels less than 20ng/ml. CONCLUSIONS: Higher levels of GP73 can be found in the serum of patients with HCC than of those without. GP73 was better than AFP for the diagnosis of early HCC. Further validation studies are needed to confirm the role of GP73 in the early detection of HCC.     

Expression of GP73, a resident Golgi membrane protein,in viral and nonviral liver disease

GP73 is a novel type II Golgi membrane protein of unknown function that is expressed in the hepatocytes of patients with adult giant-cell hepatitis (Gene 2000;249:53-65). Its expression pattern in human liver disease and the regulation of its expression in hepatocytes have not been systematically studied. The aims of the present study were to compare GP73 protein levels in viral and nonviral human liver disease and in normal livers, to identify its cellular sources, and to study the regulation of its expression in hepatoma cells in vitro. GP73 protein levels were quantitated in explant livers of patients with well-defined disease etiologies and compared with the levels in normal donor livers. GP73-expressing cells were identified immunohistochemically. GP73 expression in vitro was studied by Western blotting and immunofluorescence microscopy in HepG2 and SK-Hep-1 cells and in the HepG2-derived, hepatitis B virus (HBV)-transfected HepG2215 and HepG2T14.1 cell lines. Whole organ levels of GP73 were low in normal livers. Significant increases were found in liver disease due to viral causes (HBV, HCV) or nonviral causes (alcohol-induced liver disease, autoimmune hepatitis). In normal livers, GP73 was constitutively expressed by biliary epithelial cells but not by hepatocytes. Hepatocyte expression of GP73 was dramatically up-regulated in diseased livers, regardless of the etiology, whereas biliary epithelial cell expression did not change appreciably. GP73 was present at high levels in HepG2215 cells (a cell line that supports active HBV replication), but was absent in HepG2T14.1 cells (an HBV-transfected cell line that does not support HBV replication) and in HBV-free HepG2 cells. In SK-Hep-1 cells, GP73 expression was increased in response to interferon gamma (IFN-gamma), and inhibited by tumor necrosis factor alpha (TNF-alpha). In conclusion, increased expression of GP73 in hepatocytes appears to be a general feature of advanced liver disease, and may be regulated via distinct pathways that involve hepatotropic viruses or cytokines.     

Possible roles of Golgi protein-73 in liver diseases

Golgi protein 73 (also known as GP73 or GOLPH2) is a transmembrane glycoprotein present in the Golgi apparatus. In diseased states, GP73 is expressed by hepatocytes rather than by bile duct epithelial cells. Many studies have reported that serum GP73 (sGP73) is a marker for hepatocellular carcinoma (HCC). For HCC diagnosis, the sensitivities of sGP73 were higher than that of other markers but the specificities were lower. Considering that the concentration of GP73 is consistent with the stage of liver fibrosis and cirrhosis, some studies have implied that GP73 may be a marker for liver fibrosis and cirrhosis. Increased sGP73 levels may result from hepatic inflammatory activity. During liver inflammation, GP73 facilitates liver tissue regeneration. By summarizing the studies on GP73 in liver diseases, we wish to focus on the mechanism of GP73 in diseases.     

血清磷酯酰肌醇蛋白聚糖3与高尔基体蛋白73检测在肝细胞癌诊断中的意义

目的：评价血清磷酯酰肌醇蛋白聚糖3（gpc3）和高尔基体蛋白73（gp73）的检测对肝细胞癌（HCC）的筛查作用。方法采集70例 HCC 癌及肝硬化（肝脏再生结节）患者基线时外周血。应用 ELISA 法检测患者血清 gpc3和gp73,并与其血清AFP水平做对照。结果 HCC组gpc3、gp73测量值显著高于肝硬化组（P＜0．01）;gpc3诊断HCC的截断值＞9．3μg/L,AUC＝0．956,灵敏度为89．74％,特异度为96．77％,阳性预测值为97．2％,阴性预测值为88．2％,约登指数为0．8652,阳性似然比27．82,阴性似然比0．11,gp73诊断 HCC 的截断值＞77．68μg/L,AUC＝0．937,灵敏度为92．31％,特异度为83．87％,阳性预测值为87．8％,阴性预测值为89．7％,约登指数0．7618,阳性似然比5．72,阴性似然比0．092。结论血清gpc3与gp73水平可用于 HCC的筛查。     

Relationship between the Level of Serum Golgi Protein 73 and the Risk of Short-term Death in Patients with ALD-ACLF

Background and AimsAs a hepatocellular carcinoma biomarker, serum Golgi protein 73 (GP73) is reportedly related to inflammation. Acute-on-chronic liver failure (ACLF) is characterized by severe systemic inflammation. In this study, we aimed to explore the association between the GP73 level and short-term mortality in patients with alcohol-associated liver disease-related ACLF (ALD-ACLF).MethodsThis retrospective cohort study involved 126 Chinese adults with ALD-ACLF. Baseline serum GP73 level was measured using enzyme-linked immunosorbent assay. Patients were followed-up for 90 d and outcomes were assessed. Data were analyzed using multivariate Cox regression and piecewise linear regression analyses. The predictive value of GP73 and classic models for the short-term prognosis of participants were evaluated and compared using receiver operating characteristic curves.ResultsThe serum GP73 level was independently associated with an increased mortality risk in patients with ALD-ACLF. Compared with the lowest tertile, the highest serum GP73 level predisposed patients with ALD-ACLF to a higher mortality risk in the fully adjusted model [at 28 days: hazard ratio (HR): 4.29 (0.99–18.54), p=0.0511; at 90 days: HR: 3.52 (1.15–10.79), p=0.0276]. Further analysis revealed a positive linear association. GP73 significantly improved the accuracy of the Child-Turcotte-Pugh score, model for end-stage liver disease score, and model for end-stage liver disease-sodium score in predicting short-time prognosis of patients with ALD-ACLF.ConclusionsThe serum GP73 level is a significant predictor of the subsequent risk of death in patients with ALD-ACLF. GP73 improved the predictive value of classic prognostic scores.     

Prognostic potential of the small GTPase Ran and its methylation in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. The prognostic significance of Ran, a member of Ras superfamily, remains unclear in HCC patients.MethodsBased on The Cancer Genome Atlas (TCGA) database and Tumor Immune Estimation Resource (TIMER), we analyzed the correlations among Ran expression, promoter methylation and immune cell infiltration. We also investigated the Ran expression levels in HCC tissues and normal tissues by using quantitative real-time PCR.ResultsRan mRNA expression was significantly increased in HCC tissues compared with the normal tissues (P < 0.001). Time-dependent receiver operating characteristic (ROC) curves showed that Ran expression had predictive value of the 1-, 3- and 5-year overall survival for HCC patients, and the areas under the curves (AUC) were 0.747, 0.634 and 0.704, respectively. Cox regression analysis showed that Ran expression was an independent prognostic factor for HCC patients (HR = 1.492, 95% CI: 1.129-1.971, P = 0.005). We also found a negative relationship between Ran mRNA expression and its promoter methylation (r = -0.36, P < 0.001). High Ran expression and promoter hypomethylation predicted worse overall survival and progression-free survival (P < 0.05) and were involved in the progression of HCC. Ran expression exhibited significant correlations with immune infiltrates and prognostic immune-related genes.ConclusionsThe present study provides further insight into the prognosis of HCC, and Ran could serve as a biomarker for predicting the survival of HCC patients.     

GP73在原发性肝癌组织中的表达及意义

目的探讨高尔基体糖蛋白73(GP73)表达诊断肝癌的价值。方法采用免疫组织化学染色SP法检测60份肝癌组织、癌旁组织及24份正常肝组织中GP73表达;分析GP73表达与肝癌临床病理特征的关系。结果肝癌组织、癌旁组织GP73阳性率显著高于正常肝组织(P<0.05)。GP73表达与Child-Pugh分级呈正相关(P<0.05),在肝外转移和HBV感染中表达亦显著增强(P<0.05),与性别、年龄、AFP、肿瘤大小、肝硬化和门脉癌栓无明显相关性。结论 GP73与肝癌的发生发展有关。     

血清高尔基体蛋白73诊断肝细胞癌价值探讨

目的评价血清高尔基体蛋白73（GP73）在肝细胞癌（HCC）诊断中的价值。方法选择400例HCC、100例肝硬化（LC）、100例慢性乙型肝炎（CHB）患者和200例健康对照者（HC）,分别采用ELISA和电化学发光法检测血清GP73和甲胎蛋白（AFP）水平;采用受试者工作特征曲线下面积（ AUROC）评估两者对HCC的诊断效能。结果 HCC、CHB和LC患者血清GP73水平分别为243.6±163.1ng/ml、85.6±35.3ng/ml和81.9±36.4ng/ml,均显著高于健康对照组（78.2±33.9ng/ml,P〈0.05）,但在CHB和LC组及HC三组间无统计学差异;GP73和AFP的最佳诊断临界值分别为150.7ng/ml和19.7ng/ml,GP73的AUROC为0.846,灵敏性为69.2%,特异度为90.5%,均显著优于AFP（AUROC为0.828,灵敏性为57.6%,特异度为88.1%,P〈0.05）;GP73与AFP两者联合检测可以进一步提高对HCC的诊断准确性（AUROC为0.887,灵敏性为73.5%,特异度为88.0%）;在AFP阴性与阳性的两组HCC患者中,GP73灵敏性和特异度无显著性差异。结论血清GP73对HCC的诊断效能高于AFP,两者联合检测可以进一步提高诊断效能。