الفهرس 
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Abstract
The findings of the present study suggested that FZD7 rs2280509 polymorphism is associated with an increased risk of HCC in the Egyptian population.
FZD7, GPC3 and AFP have a complementary role in early HCC detection. FZD7 gene expression and GPC3 gene expression increase the diagnostic yield in AFP-negative HCC and have greater diagnostic sensitivity and accuracy than AFP in the diagnosis of HCC. Additionally, GPC3 and FZD7 have promising roles in the pre-clinical diagnosis of HCC.
Finally, according to the current results it can be concluded that, FZD7 gene expression has a better sensitivity than AFP for the diagnosis of HCC. Current evidence indicates that FZD7, GPC3 with AFP exhibit much better sensitivity for the diagnosis of HCC when used in combination rather than alone.
Further studies on larger samples from different geographic regions are needed to explore the association between FZD7 whether gene expression or gene polymorphism and HCC risk.
Summary
Hepatitis C virus (HCV) infection is a global health challenge; the World Health Organization (WHO) estimates that more than 71 million subjects have chronic HCV infections. HCV causes advanced fibrosis and cirrhosis and therefore it is the major causative agent of hepatocellular carcinoma (HCC), because the majority of HCC cases develop in cirrhotic livers. HCC is the most common primary malignancy of the liver and the second leading cause of cancer-related deaths worldwide. HCC is a complex biological process and is often diagnosed at advanced stages with no effective treatment options. HCC has been shown to progress in a multistep manner, although the molecular basis of HCC carcinogenesis has not been clearly identified. HCC surveillance with alpha-fetoprotein (AFP) and ultrasonography (US) has been recommended for patient with cirrhosis; however, the AFP level is insensitive for early diagnosis of the disease, and since early detection of HCC is essential, new markers with sufficient sensitivity and specificity are needed.
There are numerous protein pathways involved in the development and progression of HCC, including both stimulatory and inhibitory pathways. Among the molecular signaling pathways implicated in the pathogenesis of HCC, the Wnt/β-catenin signaling pathway is one of the most frequently activated, and is dysregulated in hepatic cancers.
The Frizzled (FZD) is a family of G protein-coupled receptor (GPCRs) proteins that serves as receptors in the Wnt signaling pathway and other signaling pathways. Of the 10 members of the Fzd family, Frizzled 7 (FZD7) is the most important Wnt receptor involved in cancer development and progression. Among all FZD receptors, FZD7 is relatively more characterized in HCC.
Glypican-3 (GPC3) is a member of heparan sulfate proteoglycans which attaches to the cell membrane and is frequently observed to be elevated in HCC. GPC3 is one of six mammalian members of the glypican family of proteoglycans. GPC3 is a highly tumor specific antigen, in addition, GPC3 promotes HCC growth and metastasis by activating the canonical Wnt signaling pathway by directly interacting with Wnts and Frizzled to promote the growth of HCC.
Single nucleotide polymorphisms (SNPs) are prominent sources of variation in human genome and serve as excellent genetic markers for constructing high genetic maps and to carry out association studies related to diseases. SNPs have been implicated in the assessment of risk, early diagnosis, prevention, treatment of diseases, and drug development.
The aim of the present study was to evaluate the validity of FZD7 and GPC3 gene expression as potential biomarkers for HCC early diagnosis, and to investigate the association between FZD7 rs2280509 polymorphism and HCC risk.
The current study was conducted on one hundred and sixty four participants (164); 50 normal individuals as normal controls, 57 patients with liver cirrhosis (LC) and 57 patients with HCC recruited from National Hepatology and Tropical Medicine Research Institute from December 2016 to April 2017.
Patients with severe heart or brain disease, or with viral disease other than HBV or HCV infection, and patients with other parts of the body or organs associated with malignant tumor were excluded from this study. Full history was taken with special reference to risk factors of liver diseases such as previous HCV exposure in surgical wards, blood transfusion, dental therapy, needle stick injury, history of HCV.
Routine laboratory investigations were carried out: AST, ALT, total bilirubin, albumin, creatinine, random blood sugar, prothrombin time, AFP, haemoglobin, WBCs count and platelet count. Genotyping of FZD7 gene polymorphism, FZD7 gene expression and GPC3 gene expression levels were determined using Real Time PCR technique.
Continuous variables were presented as mean ± standard deviation (SD) for normal distribution or median and interquartile range (IQR) for abnormal distribution, while categorical variables were presented as absolute numbers. Associations between categorical variables were tested by the calculation of Chi-Square test.
Comparison between two independent groups was done using Mann-Whitney U test. Differences between groups were measured by the calculation of one-way ANOVA. Correlations between biochemical markers and continuous variables were tested using the Spearman’s rank correlations.
Prior to association analyses, deviation from Hardy-Weinberg equilibrium (HWE) was tested for gene polymorphism using the Michael H. Court’s (2005–2008) calculator Excel-based HWE Test. Genotype and allele frequencies were compared between the disease and the control groups. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using Chi-square test to investigate the association between the genotype frequencies and HCC risk.
Sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy were calculated. The predictive values of the studied parameters for the patient’s groups were compared to control group data by ROC curve analysis. Data were expressed as area under the curve (AUC). A binary logistic regression model was used to evaluate the diagnostic capacity of the combined biomarkers.
Results revealed that:
HCC patients showed a significantly higher mean values of ALT, AST, total bilirubin, creatinine, INR and glucose (P<0.001) compared to the control group. Cirrhotic patients showed a significantly higher mean values of ALT, AST and creatinine (P<0.01), with a significantly higher mean values of total bilirubin, glucose and INR (P<0.001) compared to the control group.
The median expression values of FZD7 and GPC3 levels were significantly higher in LC group and HCC group compared to control group (P <0.001).
A highly significant positive correlation (r= 0.493, P<0.001) was found between FZD7 gene expression levels and tumor size ≥5 cm. On the other hand, Spearman’s correlation analysis revealed a significant positive correlation between FZD7 and GPC3 gene expression levels in HCC patients (r =0.344, P=0.009).
The CT genotype and T allele were significantly more prevalent in the HCC group compared to either the cirrhosis or control groups. Meanwhile, the CC genotype and C allele were significantly more prevalent in the control group, indicating that the FZD7 rs2280509 CT genotype may be a potential risk factor for HCC.
The study did not reveal any significant correlation between FZD7 gene expression and either its genotype frequency (P=0.061) or its allele frequency (P=0.051). Meanwhile the combined CT + TT genotypes had significantly lower FZD7 expression levels than the CC genotype (P=0.019).
from ROC curve analysis, the cut-off value for AFP was 21 µg/L with sensitivity 65% and specificity 73%. The cut-off value for GPC3 expression was 192 with sensitivity 77% and specificity 72%, while the cut-off value for FZD7 expression was 192 with sensitivity 86% and specificity 64%.
FZD7 gene expression, alone (AUC =0.820, sensitivity 86%) or in combination with AFP (AUC =0.845, sensitivity 95%), was better than AFP alone in the diagnosis of HCC. Meanwhile combining the two markers AFP and GPC3 expression had a better AUC of 0.853, sensitivity of 84%, and specificity of 72%.
Consequently, the combination of AFP with GPC3 or the combination of AFP with FZD7 could both increase the diagnostic capacity of AFP with greater AUC.