Evaluation of circulating Transforming growth factor-beta1, Glypican-3 and Golgi protein-73 mRNAs expression as predictive markers for hepatocellular carcinoma in Egyptian patients

Hepatocellular carcinoma (HCC) incidence is fast-growing especially in countries highly prevalent with viral hepatitis. Its poor prognosis has driven the research toward the discovery of sensitive markers for early detection. We investigated the usefulness of serum Transforming growth factor-beta1 (TGF-β1), Glypican-3 (GPC3), and Golgi protein-73 (GP73) mRNAs as early biomarkers in HCC Egyptian patients chronically infected with hepatitis C virus (HCV) in comparison with serum alpha-fetoprotein (AFP). Using semi-quantitative RT-PCR and densitometry analysis, circulating TGF-β1, GPC3, and GP73 mRNAs expressions were estimated in 15 healthy adults, 15 chronic HCV (CHC) patients and 25 HCC patients. Serum GP73 expression percentage in HCC group was significantly higher than controls (100 vs. 40 %, P ≤ 0.001) and when compared to elevated serum AFP levels (100 vs. 36 %, P ≤ 0.001). TGF-β1 and GP73 expression means were also higher in HCC patients than controls and CHC patients (P < 0.05). GPC3 expression showed higher frequency in CHC patients compared to HCC group (80 vs. 28 %, P = 0.0016). According to the study cutoffs, serum TGF-β1 and GP73 mRNAs showed 60 and 96 % sensitivities for HCC diagnosis with 100 and 95 % specificities, respectively. Furthermore, elevated GP73 mRNA expression levels in early HCC were significantly increased compared to those of TGF-β1 mRNA and to high serum AFP (92.3 vs. 53.8 and 23.1 %; P = 0.03 and 0.0004, respectively). In conclusion, circulating TGF-β1 and GP73 mRNAs could be useful biomarkers for HCV-induced HCC diagnosis. Moreover, serum GP73 mRNA is sensitive for early cancer detection than AFP and TGF-β1 mRNA. However, these results need further validation studies.     

AFP、AFU、β_2-MG、CA199联合检测对原发性肝癌的早期诊断价值

目的：探讨原发性肝癌患者血清甲胎蛋白（AFP）、а-L岩藻糖苷酶（AFU）、β2-微球蛋白（β2-MG）、糖类抗原-199（CA199）的含量及其联合检测对原发性肝癌的早期诊断价值。方法：选择56例原发性肝癌患者、60名肝炎肝硬化患者和60名健康对照作为研究对象,分别应用比值法和化学发光法、生化法检测其血清AFP、AFU、β2-MG、CA199的含量。结果：原发性肝癌患者血清AFP、AFU、β2-MG、CA199含量均显着高于肝炎肝硬化组及健康对照组,差异均有统计学意义（P均〈0.05）;联合检测AFP、AFU、β2-MG、CA199四种肿瘤标志物,其阳性率达（85.7%）明显高于AFP（53.6%）、AFU（55.4%）、β2-MG（48.2%）和CA199（42.9%）单项检测组（P均〈0.05）;且AFP、AFU、β2-MG、CA199四种肿瘤标志物联合检测的敏感性均高于单一检测指标,差异有统计学意义（P〈0.05）,但其特异性显著低于AFU、β2-MG单项检测（P〈0.05）。结论：联合检测血清AFP、AFU、β2-MG、CA199含量可以提高对原发性肝癌的阳性诊断率,对诊断及鉴别诊断原发性肝癌具有重要意义。     

Expression of cyclooxygenase-2 and transforming growth factor-beta1 in HCV-induced chronic liver disease and hepatocellular carcinoma

Cyclooxygenase-2 (COX-2) and transforming growth factor-beta1 (TGF-beta1) were modulated in a variety of viral infections, but there is a paucity of data about their role in the pathologic process of cirrhosis and/or hepatocellular carcinoma (HCC) following chronic hepatitis C virus (HCV) infection. The material of the current study included 50 cases of chronic hepatitis C (CHC) without cirrhosis, 30 cases of CHC with cirrhosis, and 30 cases of HCC with HCV admitted to the Gastroenterology and Hepatology Department of Theodor Bilharz Research Institute, Giza, Egypt. Fifteen wedge liver biopsies, taken during laparoscopic cholecystectomy, were included in the study as normal controls. Laboratory investigations, serologic markers for viral hepatitis, and serum alpha fetoprotein levels (alpha-FP) were done for all cases of the study. Immunohistochemistry using primary antibodies against both factors revealed weak to faint immunoreactivity to COX-2 and TGF-beta1 in normal hepatic tissue (< 30% and < 50% of the cells, respectively). COX-2 expression was upregulated in patients with CHC with and without cirrhosis, yet 80% of positively stained cirrhotic cases showed marked staining intensity. Higher COX-2 expression was observed in well-differentiated HCC cases (80%) with marked staining intensity (75%) compared with advanced HCC tumors (P < .001). TGF-beta1 was expressed in the hepatocytes of all cases of CHC with and without cirrhosis as well as in 67% of HCC cases. Extensive cytoplasmic expression was detected in 52%, 93.3%, and 46.6% of CHC patients without cirrhosis, patients with cirrhosis, and patients with HCC, respectively. A positive correlation was observed between hepatic expression of COX-2 and TGF-beta1 (r = 0.67, P < .05); however, no correlation was detected between the latter and grade of HCC differentiation (r = 0.33, P > .05). CONCLUSION: These findings may suggest that TGF-beta1 plays a role in hepatic cell damage following HCV infection thus stressing the usefulness of this cytokine as a prognostic marker for liver cell injury. However, COX-2 is a predictive marker for malignant transformation and has a role in the early stages of hepatocarcinogenesis, but not in the advanced stages. The combined expression of both factors in HCV-related HCC suggests their synergistic action in the pathophysiology of hepatocarcinogenesis.     

Secreted ERBB3 isoforms are serum markers for early hepatoma in patients with chronic hepatitis and cirrhosis

Most hepatocellular carcinoma (HCC) is generated from chronic hepatitis and cirrhosis. To discover new markers for early HCC in patients with chronic hepatitis and cirrhosis, we initiated our search in the interstitial fluid of tumor (TIF) via differential gel electrophoresis and antibody arrays and identified secreted ERBB3 isoforms (sERBB3). The performance of serum sERBB3 in diagnosis of HCC was analyzed using receiver operating characteristic curves (ROC). The serum sERBB3 level was significantly higher in HCC than in cirrhosis (p < 0.001) and chronic hepatitis (p < 0.001). The accuracy of serum sERBB3 in detection of HCC was further validated in two independent sets of patients. In discrimination of early HCC from chronic hepatitis or cirrhosis, serum sERBB3 had a better performance than alpha-fetoprotein (AFP) (areas under ROC [AUC]: sERBB3 vs AFP = 93.1 vs 81.0% from chronic hepatitis and 70.9 vs 62.7% from cirrhosis). Combination of sERBB3 and AFP further improved the accuracy in detection of early HCC from chronic hepatitis (AUC = 97.1%) or cirrhosis (AUC = 77.5%). Higher serum sERBB3 levels were associated with portal-vein invasion and extrahepatic metastasis of HCC (p = 0.017). Therefore, sERBB3 are serum markers for early HCC in patients with chronic hepatitis and cirrhosis.     

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.     

Serum levels of angiogenic and anti-angiogenic factors: their prognostic relevance in locally advanced hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prototype tumor wherein angiogenesis plays a vital role in its progression. The role of VEGF, a major angiogenic factor in HCC is known; however, the role of anti-angiogenic factors simultaneously with the angiogenic factors has not been studied before. Hence, in this study, the serum levels of major angiogenic [Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 (Ang-2)] and anti-angiogenic (endostatin, angiostatin) factors were analyzed and correlated with clinico-radiological features and with outcome. A total of 150 patients (50 HCC, 50 cirrhosis and 50 chronic hepatitis) and 50 healthy controls were enrolled in this study. Serum levels of VEGF, Ang-2, endostatin, and angiostatin were estimated by enzyme-linked immunosorbent assay. HCC shows significantly elevated serum levels of angiogenic factors VEGF and Ang-2 and of anti-angiogenic factors endostatin and angiostatin. ROC curve analysis for serum VEGF yielded an optimal cut-off value of 225.14 pg/ml, with a sensitivity of 78 % and specificity of 84.7 % for a diagnosis of HCC and its distinction from other group. Using this value, the univariate and multivariate analysis revealed significantly poor outcome in patients with higher levels of serum VEGF (p = 0.009). Combinatorial analysis revealed that patients with higher levels of both angiogenic and anti-angiogenic factors showed poor outcome. Serum VEGF correlates with poor survival of HCC patients and, therefore, serves as a non-invasive biomarker of poor prognosis. Moreover, elevated levels of anti-angiogenic factors occur endogenously in HCC patients.     

The expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in peripheral blood of patients with HCC and lung cancer

There are different views of how the immune system participates in the reaction to cancer. Here, we evaluated expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in patients with hepatocellular carcinoma (HCC) and lung cancer to explore tumor immunity. Our analysis showed that levels of HSP70 and SPAG9 antibody were significantly higher in the serum of lung cancer and HCC patients than in the serum of healthy subjects (P < 0.001), but there were no differences in levels of HSP70 antibody in patients and controls. Levels of serum SPAG9 antibody in newly diagnosed lung cancer patients were significantly higher than in treated lung cancer patients (P < 0.05), but there were no differences in levels of HSP70 or HSP70 antibody. Levels of serum HSP70 and SPAG9 antibody, but not HSP70 antibody, were also higher in hepatitis/cirrhosis patients than in healthy subjects (P = 0.005, P < 0.001). Levels of serum SPAG9 antibody were significantly higher in HCC patients than in hepatitis/cirrhosis patients, but there were no differences in HSP70 or HSP70 antibody levels. Finally, levels of serum HSP70 and SPAG9 antibody were significantly higher in HCC patients than in lung cancer patients (P < 0.05, P < 0.001). These results indicate that cancer-testis antigen SPAG9 induces a strong humoral immune response in cancer patients but HSP70 does not. These results show that SPAG9 has potential as a tumor-specific biomarker.     

Evaluation of serum alpha fetoprotein-L3 as an accuracy novel biomarker for the early diagnosis of hepatocellular carcinoma in Egyptian patients

BackgroundMost Hepatocellular Carcinomas (HCCs) are diagnosed at an advanced stage. However, HCC early diagnosis is complicated by the coexistence of inflammation and cirrhosis. The unsatisfactory sensitivity and specificity of Alpha-fetoprotien (AFP) for screening of early-stage HCC paved the way for new novel biomarkers to complement AFP such as AFP-L3. The aim of this study was the Evaluation of alpha fetoprotein-L3 (AFP-L3) as earlier marker in diagnosis of hepatocellular carcinoma in Egyptian patients. This study was conducted on 80 patients categorized into 2 groups; group 2 (40 patients with chronic active hepatitis) and group 3 (40 patients with HCC). HCC diagnosis was done by clinical, triphasic CT and positive US for focal lesion, in addition to 20 healthy individuals as controls (group 1).ResultsThe median range of AFP and AFP-L3 were highly statistically significant difference between HCC group and other groups [p < 0.001]. In this study ALT, AST, Total & direct bilirubin and albumin results showed highly significant differences between HCC group and other groups. Serum AFP-L3 shows sensitivity 100%, specificity 100%, positive predictive value 100% and negative predictive value 100% with AUC = 1 in HCC cases.ConclusionSerum AFP-L3 may serve as a diagnostic biomarker for the detection of early stage of HCC and show higher sensitivity than AFP.     

DEC1-mRNA在肝癌患者血清中的表达和手术预后的关系

目的:研究DEC1在肝癌(hepatocellular carcinoma,HCC)患者血清中的表达水平及其临床意义。方法:收集60例HCC患者,30例正常人,30例慢性肝炎患者,30例慢性肝硬化患者血清标本。采用实时荧光定量RT-PCR(RT-PCR)检测血清DEC1的表达情况。分析血清DEC1的表达水平与AFP相关性。探讨血清DEC1的表达水平与HCC手术预后的关系。结果:肝癌患者血清DEC1明显高于正常人、慢性肝炎和慢性肝硬化患者(P0.05)。皮尔森相关分析结果显示HCC患者血清DEC1的表达与血清AFP水平是呈正相关的。Kaplan-Meier结果显示血清DEC1低表达组术后的复发转移率显著低于高表达组,其术后生存率也显著高于高表达组(P0.01)。结论:DEC1在肝癌者血清中高表达,不仅可能成为HCC诊断的标记物,且其血清表达水平也有助于HCC临床预后的评估。     

血清VEGF、miR-122、GDF15及TK1在乙肝相关性肝病表达水平及其临床意义

摘要 目的：分析不同类型乙肝相关性肝病患者血清血管内皮生长因子（VEGF）、microRNA-122（miR-122）、生长分化因子15（GDF-15）及胸苷激酶1（TK1）的表达差异变化及其临床意义。方法：随机选取2020年1月-2022年6月在我院消化内科收治的不同类型乙肝相关性肝病患者135例作为研究组。其中根据乙型肝炎诊断标准分为慢性乙型肝炎组（CHB）76例,乙型肝炎肝硬化组（LC）57例,乙型肝炎肝癌组（HCC）78例,选取同时期在本院进行体检的健康受检者96例作为健康组。比较各组的基本资料、检测研究对象血清VEGF、miR-122、GDF15、TK1、HBV DNA载量水平和AFP表达水平。采用Pearson相关性检验分析乙肝相关性肝病患者血清VEGF、miR-122、GDF15及TK1表达水平与HBV DNA载量水平的相关性,采用受试者工作特征曲线（ROC）诊断LC和HCC的价值。结果：CHB 组、LC 组 和HCC组VEGF、TK1表达水平显著高于健康组,差异具有统计学意义（P<0.01）,CHB 组、LC 组 和HCC组组间两两比较,差异具有统计学意义（P<0.01）。CHB 组、LC 组 和HCC组miR-122表达水平显著低于健康组,差异具有统计学意义（P<0.01）,CHB 组、LC 组 和HCC组组间两两比较,差异具有统计学意义（P<0.01）。LC 组 和HCC组GDF15表达水平显著高于 CHB 组和健康组,差异具有统计学意义（P<0.01）,CHB 组和健康组组间比较差异无统计学意义（P>0.05）。CHB 组、LC 组 和HCC组HBV DNA载量水平显著高于健康组,差异具有统计学意义（P<0.01）,LC 组 和HCC组组间比较,差异无统计学意义（P>0.05）。LC 组 和HCC组AFP表达水平显著高于 CHB 组和健康组,差异具有统计学意义（P<0.01）,LC 组 和HCC组、CHB 组和健康组组间比较差异无统计学意义（P>0.05）。CHB组、LC+HCC组血清VEGF、miR-122、GDF15及TK1水平与HBV DNA载量水平具有相关性（P<0.01或P<0.05）。血清VEGF、miR-122、GDF15及TK1单独检测诊断LC和HCC的曲线下面积（AUC）为0.695、0.783、0.743及0.7687,四项指标联合检测诊断LC和HCC的AUC为0.839,敏感度为84.21,特异度为83.25。结论：血清VEGF、miR-122、GDF15及TK1在各类型乙肝相关性肝病中表达水平存在差异,血清VEGF、miR-122、GDF15及TK1联合检测诊断LC和HCC具有临床价值。     

Identification of Serum Monocyte Chemoattractant Protein-1 and Prolactin as Potential Tumor Markers in Hepatocellular Carcinoma

Early diagnosis of hepatocellullar carcinoma (HCC) remains a challenge. The current practice of serum alpha-fetoprotein (AFP) measurement is inadequate. Here we utilized a proteomic approach to identify novel serum biomarkers for distinguishing HCC patients from non-cancer controls. We profiled the serum proteins in a group of 58 resectable HCC patients and 11 non-HCC chronic hepatitis B (HBV) carrier samples from the Singapore General Hospital (SGH) using the RayBio® L-Series 507 Antibody Array and found 113 serum markers that were significantly modulated between HCC and control groups. Selected potential biomarkers from this list were quantified using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA) array in an expanded SGH cohort (126 resectable HCC patients and 115 non-HCC chronic HBV carriers (NC group)), confirming that serum prolactin and monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in HCC patients. This finding of serum MCP-1 elevation in HCC patients was validated in a separate cohort of serum samples from the Mochtar Riady Institute for Nanotechnology, Indonesia (98 resectable HCC, 101 chronic hepatitis B patients and 100 asymptomatic HBV/HCV carriers) by sandwich ELISA. MCP-1 and prolactin levels were found to correlate with AFP, while MCP-1 also correlated with disease stage. Subsequent receiver operating characteristic (ROC) analysis of AFP, prolactin and MCP-1 in the SGH cohort and comparing their area under the ROC curve (AUC) indicated that neither prolactin nor MCP-1 on their own performed better than AFP. However, the combination of AFP+MCP-1 (AUC, 0.974) had significantly superior discriminative ability than AFP alone (AUC, 0.942; p<0.001). In conclusion, prolactin and MCP-1 are overexpressed in HCC and are conveniently quantifiable in patients’ sera by ELISA. MCP-1 appears to be a promising complementary biomarker for HCC diagnosis and this MCP-1+AFP model should be further evaluated as potential biomarker on a larger scale in patients at-risk of HCC.     

A pilot study of salivary N-glycome in HBV-induced chronic hepatitis,cirrhosis, and hepatocellular carcinoma

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to chronic liver disease and put people at high risk of death from cirrhosis of the liver and liver cancer. However, little is known about the correlation of salivary N-linked glycans related to HBV-infected liver diseases. Here we investigated N-linked glycome in saliva from 200 subjects (50 healthy volunteers (HV), 40 HBV-infected patients (HB), 50 cirrhosis patients (HC), and 60 hepatocellular carcinoma patients (HCC) using MALDI-TOF/TOF-MS. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench program. A total of 40, 47, 29, and 33 N-glycan peaks were identified and annotated from HV, HB, HC, and HCC groups, respectively. There were 15 N-glycan peaks (e.g., m/z 1647.587, 1688.613 and 2101.755) were present in all groups. Three N-glycan peaks (m/z 2596.925, 2756.962, and 2921.031) were unique in HV group, 2 N-glycan peaks (m/z 1898.676 and 1971.692) were unique in HB group, 5 N-glycan peaks (m/z 1954.677, 2507.914, 2580.930, 2637.952, and 3092.120) were unique in HC group, and 3 N-glycan peaks (m/z 2240.830, 2507.914, and 3931.338) were unique in HCC group. The proportion of fucosylated N-glycans was apparently increased in the HCC group (84.8%) than in any other group (73.1% ± 0.01), however, the proportion of sialylated N-glycans was decreased in HCC group (12.1%) than in any other group (17.23% ± 0.003). Our data provide pivotal information to distinguish between HBV-associated hepatitis, cirrhosis and HCC, and facilitate the discovery of biomarkers for HCC during its early stages based on precise alterations of N-linked glycans in saliva.     

Cancer-associated carbohydrate antigens as potential biomarkers for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ~60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.     

The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis

Background

An isoleucine>methionine mutation at position 148 in the PNPLA3 gene (p.I148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. Here, we studied whether the PNPLA3 rs738409 polymorphism also affects predisposition to hepatocellular carcinoma (HCC).

Methods

We compared distributions of PNPLA3 genotypes in 80 and 81 Caucasian patients with alcoholic and hepatitis C virus (HCV)-associated HCC to 80 and 81 age- and sex-matched patients with alcohol-related and HCV-related cirrhosis without HCC, respectively. PNPLA3 genotypes in 190 healthy individuals from the same population served as reference. Potential confounders obesity, diabetes, HCV genotype and HBV co-infection were controlled by univariate and multivariate logistic regression with forward variable selection.

Results

PNPLA3 genotypes were in Hardy-Weinberg equilibrium for all study groups. The frequency of the 148M allele was significantly (p<0.001) increased in alcoholic cirrhosis with (53.7%) and without HCC (36.2%) but was not different between healthy controls (22.9%) and patients with cirrhosis (25.3%; p = 0.545) and HCC (30.2%; p = 0.071) due to hepatitis C. HCC risk was highest in 148M/M homozygous patients with alcoholic liver disease (odds ratio (OR) 16.8 versus healthy controls; 95% confidence interval (CI) 6.68–42.43, p<0.001). Finally, multivariate regression confirmed 148M/M homozygosity (OR 2.8; 95%-CI: 1.24–6.42; p = 0.013) as HCC risk factor in alcoholic cirrhosis. In HCV-related cirrhosis only HCV genotype 1 was confirmed as a HCC risk factor (OR 4.2; 95%-CI: 1.50–11.52; p = 0.006).

Conclusion

The PNPLA3 148M variant is a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects are negligible in patients with cirrhosis due to HCV. This polymorphism provides an useful tool to identify individuals with particularly high HCC risk in patients with alcoholic liver disease that should be taken into account in future HCC prevention studies.     

Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.     

Profiles of serum microRNAs; miR-125b-5p and miR223-3p serve as novel biomarkers for HBV-positive hepatocellular carcinoma

Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions including cancer. Certain microRNAs (miRNAs) have been shown early diagnostic potential for many types of cancer. The objective of this study was to investigate the potential of certain serum/plasma miRNAs as novel non-invasive biomarkers for early diagnosis of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). For this reason, the expression levels of 24 miRNA (let-7c, miR-92a-3p, 423-5p, 150-5p, 223-3p, 125b-5p, 342-3p, miR-206, 122-5p, 375, 223-5p, 10a-5p, 23b-5p, 99a-5p, 23a-5p, 10a-3p, 122-3p, 125b-1-3p, 23b-3p, 125b-2-3p, 23a-3p, 92a-1-5p, 92a-2-5p, 99a-3p) were analyzed in plasma of patients with chronic hepatitis B, HBV-positive cirrhosis and HBV-positive HCC and compared with control group samples. Totally 94 plasma samples; 28 control and 66 patient plasma (24 CHB, 22 HBV-positive cirrhosis, 20 HBV-positive HCC) and were included in this study. The expression levels of 24 miRNAs were detected for all control and patient group plasma samples by qRT-PCR using BioMark? 96.96 Dynamic Array (Fluidigm Corporation) system. The expression levels of miR-125b-5p were detected 2.85 fold, 2.46 fold and 1.89 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) up regulated in CHB, HBV-positive cirrhosis and HBV-positive HCC, respectively when compared versus control group individually by Mann–Whitney U test. The expression levels of miR-223-3p were detected 5.55 fold, 13.88 fold and 12.65 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) down regulated in same comparisons. When all groups were compared versus control group by one-way ANOVA test, the expression levels of miR-223-3p were also found statistically significant (p < 0.05). Although not statistically significant, miR-125b-5p tended to be upregulated. (p = 0.07192). These results significantly imply that miR-125b-5p and miR223-3p could be used as novel non-invasive biomarkers of HBV-positive HCC in very early, even at CHB stage of liver disease.     

The diagnostic value of serum DSA-TRF in hepatocellular carcinoma

TRF is a glycoprotein mainly secreted by hepatocytes, The aim of this study was to explore the diagnostic value of aberrant glycosylated serum transferrin (TRF) especially containing multi-antennary glycans in hepatocellular carcinoma (HCC).A total of 581 subjects including HCC patients, liver cirrhosis (LC) patients, chronic hepatitis (CHB) patients and healthy controls (HC) were recruited. All the subjects were randomly assigned to training group (n?=?411) and validation group (n?=?170). We firstly analyzed the serum protein N-glycome profiling of HCC, LC, and HC by DNA sequencer–assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) technology. We established a lectin-antibody sandwich ELISA (Lectin-ELISA) method to detect multi-antennary glycans-contained TRF (DSA-TRF) in serum, in which Datura stramonium Agglutinin (DSA) was used for specific recognition. Levels of serum DSA-TRF and TRF were analyzed respectively. The diagnostic efficacies of DSA-TRF and TRF of differentiating HCC patients from CHB, LC patients and HC within the training group were evaluated using receiver operating characteristic (ROC) curve and tested in the validation group.The result found that in training group, serum TRF and DSA-TRF levels differed significantly between HCC (1.86?±?0.50, g/L, 0.285?±?0.06), CHB?+?LC (2.39?±?0.74, g/L, 0.189?±?0.07) and HC (1.92?±?0.69, g/L, 0.249?±?0.09) (HCC vs. CHB?+?LC, P?<?0.001; HCC vs. HC, P?<?0.001; CHB?+?LC vs. HC, P?<?0.001). The area under the ROC curve (AUC) of DSA-TRF was significantly superior to AFP (0.880, 95%CI: 0.834–0.925 vs. 0.776, 95%CI: 0.725–0.827, P?=?0.003) in differentiating HCC from CHB?+?LC. The AUC of diagnostic model GlycoTRF1 (0.981, 95%CI: 0.969–0.993) was higher than DSA-TRF and AFP alone (P<0.001) in differentiating HCC from CHB?+?LC, which was verified in validation group.The results indicated that the serum DSA-TRF might serve as a potential glycan biomarker for distinguishing HCC from CHB and LC.

     

Diagnostic Evaluation of Des-Gamma-Carboxy Prothrombin versus α-Fetoprotein for Hepatitis B Virus-Related Hepatocellular Carcinoma in China: A Large-Scale,Multicentre Study

An efficient serum marker for hepatocellular carcinoma (HCC) is currently lacking and requires intensive exploration. We aimed to evaluate the performance of des-gamma-carboxy prothrombin (DCP) for identifying hepatitis B virus-related HCC in a large, multicentre study in China. A total of 1034 subjects in three cohorts (A, B, and C) including HCC and various non-HCC controls were enrolled from 4 academic medical centers in China from January 2011 to February 2014. Blind parallel detections were conducted for DCP and AFP. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic efficacies. In cohort A, which comprised 521 subjects, including patients with HCC, liver metastasis, liver cirrhosis (LC), and liver hemangiomas as well as healthy controls (HCs), the accuracy of DCP for distinguishing HCC from various controls was 6.2–9.7% higher than that of AFP. In cohort B, which comprised 447 subjects, including patients with HCC, LC, and chronic hepatitis B as well as HC, the accuracy of DCP was further elevated (12.3–20.67% higher than that of AFP). The superiority of DCP to AFP was more profound in the surveillance of early HCC [AUC 0.837 (95% CI: 0.771–0.903) vs. 0.650 (0.555–0.745)] and AFP-negative HCC [AUC: 0.856 (0.798–0.914)] and in discriminating HCC from LC (accuracy: 92.9% vs.64.71%). Higher DCP levels were associated with worse clinical behaviors and shorter disease-free survival. DCP not only is complementary to AFP in identifying AFP-negative HCC and in excluding AFP-positive non-HCC (liver cirrhosis), but also demonstrates improved performance in HCC surveillance, early diagnosis, treatment response and recurrence monitoring in the HBV-related population.     

Estimation of Copper and Iron Burden in Biological Samples of Various Stages of Hepatitis C and Liver Cirrhosis Patients

There is accumulative evidence that the metabolism of iron (Fe) and copper (Cu) is altered in human due to infections, indicating that both elements have roles in pathogenesis and progress of viral diseases. In the present study, the correlation of Cu and Fe was evaluate in biological samples (serum and scalp hair) of hepatitis C (hepatitis C virus (HCV)) patients of both genders at different stages. For comparative study, the scalp hair and serum samples of healthy individuals of same age group (30–50 years) and socioeconomic status were collected. The biological samples were analyzed for Fe and Cu by atomic absorption spectroscopy after microwave-assisted acid digestion. The validity and accuracy of methodology were checked by certified reference materials of same matrixes. The levels of Cu and Fe in biological samples were enhanced in hepatic disorder patients, including acute (after diagnosis test, anti-HCV sero-positive) hepatic fibrosis and liver cirrhosis as compared to healthy referents. The difference was significant (p?<?0.01) in the case of liver cirrhotic patients. It was observed that the data of Cu and Fe in referents and patients of both genders had normal distributions. The inter-elemental correlation (r) among Cu vs Fe in serum and scalp hair samples of referents and patients were not significant in both genders (p?>?0.1) except in the first stage of HCV (p?<?0.1). It was concluded that the increase of Cu and Fe content in human body seems to contribute to the development of cirrhosis in patients with viral hepatitis C.