Glypican-3, Vascular Endothelial Growth Factor and Golgi Protein-73 for Differentiation between Liver Cirrhosis and Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) accounts for more than 80% of primary liver cancers. Moreover, in the next 10 years, more than one million patients are expected to die from liver cancer as estimated by the World Health Organization. The aim of the present study is to evaluate the clinical utility of using Glypican (GPC3), Vascular endothelial growth factor (VEGF) and Golgi protein 73 (GP73) in serum by Enzyme-Linked Immunosorbent Assay (ELISA) and by Real-Time Polymerase Chain Reaction (RT-PCR), as diagnostic markers to differentiate HCC from cirrhotic liver disease. METHODS: A total of 50 patients with histologically-proven HCC, 50 liver cirrhosis patients and 20 healthy volunteers as controls were enrolled in this study, blood samples were obtained from each patient. Expression of the studied biomarkers was evaluated by ELISA and Real-Time PCR. RESULTS: Statistical analysis of RT-PCR results showed that the expression of GPC3, VEGF and GP73 in serum of patients with HCC was significant (P value < 0.001, 0.01, and < 0.001) respectively and increased when compared to the cirrhotic group. Furthermore, the serum protein levels of GPC3 and VEGF in HCC and cirrhotic patients were significant when compared to the control group. While no significance was found between HCC and cirrhotic group. The serum protein level of GP73 was significantly increased in HCC and cirrhosis groups  compared to the control group (P value < 0.001). Moreover, a significant increase was  evident in HCC group compared to cirrhotic group (P value < 0.001). The results of the present study showed that the combination of VEGF and  GP73 could  discriminate HCC from cirrhosis. CONCLUSION: GPC3, VEGF and GP73 are reliable biomarkers for diagnosis of  HCC. The serum level of GP73 is a potential screening marker for discriminating HCC from liver cirrhosis.

Molecular Detection of Genetic Susceptibility to Bladder Cancer in Egyptian Patients.

OBJECTIVE: Genome-wide association studies (GWAS) have identified a number of genetic variants associated with the susceptibility of bladder cancer (BC) in European and Chinese populations. Here, we assessed the association of two of these variants, rs9642880 and rs710521 in an Egyptian patients and also examined the expression of c-Myc.The basis was due to the absence of studies on Egyptian patients to determine the association between rs9642880& rs710521 and bladder cancer risk, particularly due to the known role of the variant (rs9642880) in the Progression and development of bladder cancer. METHODS: Urine samples were collected from onehundred and fiftybladder cancer patients under particular standards and fifty healthy controls. Genomic DNA was extracted,  rs9642880 G>T and rs710521 A>G polymorphisms were amplified, assessed via restriction fragment length polymorphism(RFLP) and sequenced. Urine retrieved results were compared to the histopathological diagnosis of tissue biopsies and to the results of C-Myc immunohistochemistry. Data were statistically analyzed using Microsoft Excel 2016, association between significant genotypes of the two studied variables and bladder cancer risk was performed. RESULTS: We found that the TT genotype of rs9642880 G>T was strongly associated with the risk of bladder cancer, andfor rs710521 A>G, AG genotype was also identified to has an association with bladder cancer risk.All 150 tumor sections showed positive immunoreactivity for c-Myc in the nucleus and the cytoplasm. CONCLUSION: Identifying the association to risk of bladder cancer using genetic analysis will help in the early detection of the disease.

A Novel Cell-based In vitro Assay for Antiviral Activity of Interferons α, ß, and γ by qPCR of MxA Gene Expression.

BACKGROUND: Human MxA gene is related to the class of interferon (IFN)-stimulated genes (ISGs) that plays a role in antiviral resistance. OBJECTIVE: Implementation of standard curves obtained from designing a procedure for data processing in relative qPCR between MxA expression and interferon's antiviral activity (IU/ml). These standard curves can be used to detect the antiviral activity of any new compound rapidly and safely. METHODS: To detect the optimum incubation time for maximum MxA gene expression in human peripheral blood mononuclear cells (PBMC), the isolated human PBMCs (1x106 cells) were incubated with a concentration of 1000 IU/ml of each IFN at different time intervals; 2 h, 4 h, 6 h, and 24 h post-treatment. A standard curve was performed for each IFN (α, ß, and γ) at different concentrations (250, 500, 750, 1000, 1500, and 2000 IU/ml). RESULTS: As observed at 4 h incubation time of 1000 IU/ml concentration, IFN-γ provided a higher expression of MxA compared to IFN-α and IFN-ß. Correlation analyses between IFN-α and IFN-ß, IFN-ß and IFN-γ were non-significant. However, there was a significant correlation between IFN-α and IFN-γ (p<0.01). Receiver operator characteristic (ROC) analysis revealed that cut-off values of IFN- γ, IFN-ß, and IFN-α were 58.14 > 7.31 and > 3.33, respectively. CONCLUSIONS: The relative expression of MxA is a biomarker for IFN-α, ß, and γ, especially IFN-α. It has compiled and validated a standard curve-based protocol for PCR data processing. It shows that the standard curve is an easy alternative tool to assess antiviral activity. We revised all patents relating to the antiviral assays of the used interferons.

Evaluation of Osteopontin as a Biomarker in Hepatocellular Carcinomas in Egyptian Patients with Chronic HCV Cirrhosis

Background: Hepatocellular carcinoma (HCC) is a high incidence disease in Egypt with a poor prognosis and survival. Biomarkers are important for diagnosis of HCC at an early stage. Osteopontin (OPN), a glycoprotein secreted by macrophages, osteoblasts, and T cells, is also highly expressed in a variety of tumors, such as examples in the breast, colon, and stomach. The present study aimed to correlate the serum level of OPN in HCV-positive hepatocellular carcinoma patients, with OPN expression in tumor and non-tumor liver tissues in order to identify its efficacy as a biomarker for diagnosis. Material and Methods: Out of total of 146 patients, 80 were selected for inclusion in the study. Blood samples as well as specimens of tumor and non-tumor liver tissue were collected. In addition, blood samples from 20 healthy volunteers were obtained as controls. Serum OPN and alpha-fetoprotein (AFP) were evaluated by ELISA for HCC and control groups. OPN and AFP gene expression were examined by real-time PCR, after homogenization and DNA extraction from serum samples and liver tissues. Results: It was found that serum OPN levels were significantly higher in the HCC group compared to normal group (P=0.009), with a strong positive correlation with AFP expression. However, there was no significant difference between OPN expression in tumor and non-tumor liver tissue. Conclusion: Serum OPN is highly suggested to be a professional candidate for HCC early diagnosis, with a diagnostic ability and accuracy equal or higher than for AFP.

Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

Objective: To investigate the expression of TTF-1, RAGE, GLUT1 and SOX2 in HCV-associated HCCs and in surrounding non-tumorous liver tissue. Material and Methods: Tissue material from partial hepatectomy cases for HCC along with corresponding serum samples and 30 control serum samples from healthy volunteers were studied. Biopsies were classified into: non-tumor hepatic tissue (36 sections); HCC (33 sections) and liver cell dysplasia (LCD) (15 sections). All cases were positive for HCV. Immunohistochemistry (IHC), gene extraction and quantitative real-time reverse-transcription assays (qRT-PCR) were applied. Results: By IHC, LCD and HCC showed significantly high percentages of positive cases with all markers. SOX2 showed significant increase with higher HCC grades, while RAGE demonstrated an inverse relation and GLUT-1 and TTF-1 lacked any correlation. In nontumorous-HCV tissue, we found significantly high TTF-1, low RAGE and negative SOX2 expression. RAGE, GLUT-1 and SOX2 show non-significant elevation positivity in high grade HCV compared to low grade lesions. TTF-1, RAGE and SOX2 exhibited low expression in cirrhosis compared to fibrosis. Biochemical studies on serum and tissue extracts revealed significant down-regulation of RAGE, GLUT-1 and SOX2 genes, as well as significant up-regulation of the TTF-1 gene in HCC cases compared to controls. All studied genes show significant correlation with HCC grade. In non-tumor tissue, only TTF-1 gene expression had a significant correlation with the fibrosis score. Conclusion: Higher expression of TTF-1, RAGE, GLUT-1 and SOX2 in HCC and dysplasia compared to non-tumor tissues indicates up-regulation of these markers as early events during the development of HCV-associated HCC.

Evaluation of Mir-224, Mir-215 and Mir-143 as Serum Biomarkers for HCV Associated Hepatocellular Carcinoma

HCV induced hepatitis and hepatocellular carcinoma as its sequel are major health problems world-wide and especially in Egypt. For diagnosis and during treatment of liver diseases, liver functions are monitored through determination of serum levels of liver enzymes and α-fetoprotein although the obtained information is generally not sufficient for either early detection of hepatic insult or effective follow up of therapeutic effects. More sensitive biomarkers may help to achieve these goals. MiRNAs are small non-coding RNAs that have an important role in gene expression and regulation. Many, such as miR-224, miR-215, miR-143 are correlated with tumor appearance and with the degree of fibrosis in lung, breast and colon cancer. This study was performed to estimate the level of these miRNAs in serum of patients with HCV-associated hepatitis and HCC in relation to grade of hepatitis, stage of fibrosis and differentiation of tumor tissue. In addition, correlations between serological and tissue levels were assessed. A total of 80 patients were examined, out of which 50 were included in the study. Blood samples and tissue specimens from malignant tumor and corresponding non-tumor tissue of HCV hepatitis patients were collected. Blood samples from 20 healthy volunteers were also obtained as controls. It was found that miRNAs profiles differed in HCC patients compared to controls and HCV-associated hepatitis cases. Distinction of tumor grade and fibrosis stage of patients as well as between different grades of tumor differentiation proved possible, making miRNAs promising biomarkers for diagnosis and assessment of treatment response of HCC patients.

Differential Expression of Glypican-3 and Insulin­Like Growth Factor-II mRNAs and Alpha-Fetoprotein and Ki-67 Markers in HCV Related Hepatocellular Carcinomas In Egyptian Patients

Background: Increasing evidence indicates that in hepatocellular carcinomas (HCCs) abnormal gene expression, for example of glypican-3 (GPC-3) and insulin-like growth factor-II (IGF-II), are associated with the occurrence and progression of HCC. The objective of this study was to evaluate the differential expression of GPC-3 and IGF-II mRNAs in HCC tissues with a background of chronic hepatitis C virus (HCV) genotype 4 cirrhosis, in relation to Ki-67 and alpha-feto protein (AFP) tissue markers. Methods: One hundred and five patients with HCCs who had undergone hepatectomy, were included, after obtaining informed consent. Total RNA was extracted from malignant and corresponding peri-malignant liver tissues, and GPC-3 and IGF-II mRNAs in addition to beta-actin mRNA as an internal control, were evaluated in all samples by reverse transcriptase-polymerase chain reactions (RT-PCR). Routine histopathological diagnosis as well as immunohistochemical (IHC) staining using monoclonal antibodies for Ki-67 and AFP were also performed. Result: Expression of GPC-3 mRNA was positive in all HCC malignant tissue, with overexpression in 86/105 (81.9%); in respect to the grade of the tumor (1-3 grades), while in peri-malignant tissue it was over expressed only in 20/105 (19%). The IGF-II mRNA was over expressed in only 10/105 (9.5%) malignant and peri-malignant samples. AFP was expressed in 33.3% of malignant samples but absent in peri-malignant tissues. Ki-67 expression was significantly increased in malignant compared to peri-malignant tissue. Conclusion: GPC-3 and IGF II mRNAs may be good molecular markers for HCC, especially with a background of cirrhosis due to chronic HCV infection. Significant correlations were noted with the pattern of AFP and Ki-67 expression.