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ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 1  |  Page : 112-117

Genetic polymorphism of epidermal growth factor gene as a predictor of hepatocellular carcinoma in hepatitis C cirrhotic patients


1 Department of Internal Medicine, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Department of Liver and Hepatobiliary Surgery, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt
4 Department of Pathology, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia, Egypt
5 Department of Hepatology, Mahlla Teaching Liver Hospital, Gharbeya, Egypt

Date of Submission17-May-2019
Date of Decision14-Jun-2019
Date of Acceptance19-Jun-2019
Date of Web Publication27-Mar-2021

Correspondence Address:
Mohamed M. M. Elhoseeny
Mahlla, Gharbeya
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_179_19

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  Abstract 


Objectives
To investigate the correlation between the epidermal growth factor (EGF) polymorphism and the risk of hepatocellular carcinoma (HCC) in hepatitis C viral (HCV)-related cirrhotic patients as well as its relation to EGF protein expression in HCC tissue.
Background
In Egypt, the incidence of HCC is ∼4.7% in chronic liver disease patients because of HCV infection. EGF plays an important role in hepatocyte regeneration and also plays a role in malignant transformation. A functional polymorphism in EGF was identified recently as the G/G genotype was associated with a higher risk of HCC and increased EGF expression in serum and liver tissue from HCC patients.
Patients and methods
This case–control study was carried out on 100 patients in the HCC clinic of the National Liver Institute, Menoufia University. Routine investigations and EGF genotyping were performed in 50 HCC patients (group I) subdivided into 25 patients with resectable HCC (subgroup IA) and 25 patients with advanced unrespectable HCC (subgroup IB). Twenty-five cirrhotic HCV patients did not have HCC (group II). There were a total of 25 healthy individuals (group III). Immunohistochemical detection of EGF was performed in HCC tissue biopsy from patients who underwent surgical resection.
Results
The GG genotype was associated with a significantly increased risk of HCC compared with the AG and AA genotypes (P = 0.031) in the cirrhotic group. The G allele led to a highly significant risk of HCC compared with allele A in recessive model GG versus AG + AA (P = 0.036) rather than in the dominant model GG + AG versus AA (P = 0.66). EGF expression in tumor tissue was statistically different only between GG and AG genotypes in the resectable HCC group (group IA) (P = 0.032).
Conclusion
The EGF gene polymorphism (GG genotype) led to a significant risk of HCC development in cirrhotic patients more than the AG and AA genotypes.

Keywords: epidermal growth factor gene polymorphism, hepatitis C virus, hepatocellular carcinoma


How to cite this article:
Baghdadi I, Abu Ella KA, Elsharawy A, Elshayb E, El-Rebey HS, Elhoseeny MM. Genetic polymorphism of epidermal growth factor gene as a predictor of hepatocellular carcinoma in hepatitis C cirrhotic patients. Menoufia Med J 2021;34:112-7

How to cite this URL:
Baghdadi I, Abu Ella KA, Elsharawy A, Elshayb E, El-Rebey HS, Elhoseeny MM. Genetic polymorphism of epidermal growth factor gene as a predictor of hepatocellular carcinoma in hepatitis C cirrhotic patients. Menoufia Med J [serial online] 2021 [cited 2021 Dec 4];34:112-7. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/112/312008




  Introduction Top


Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer deaths worldwide [1]. HCC represents 70.48% of liver malignancies. Its occurrence is because of cirrhosis, which in turn is a complication of hepatitis C virus (HCV). Egypt has a very high incidence of HCV, almost six million individuals, and HCV viremia is reported in 7.3% [2]. The risk factors vary widely in different geographic regions worldwide [3]. HCC has multiple etiologies such as chronic hepatitis B and C infections, alcoholism, and many other etiological factors [4]. Abnormal gene expression, epigenetic changes, and chromosomal aberrations play an important role in the development of HCC [5]. Recently, many signaling pathways especially pathways that regulate the physiological processes such as tumor cells growth, differentiation, migration, apoptosis, and angiogenesis have been studied in the era of HCC development such as the epidermal growth factor (EGF) signal pathway [6]. EGF was found to facilitate DNA synthesis, regeneration, tumor growth, and progression of HCC cells, and bind with EGFR as the potential connection between inflammation and HCC and one of therapeutic opportunities [7]. EGF is a mitogen for hepatocytes and plays a critical role in liver tissue regeneration [8]. A functional polymorphism in EGF at position 61 (A>G) was identified recently with the G/G genotype with higher gene expression compared with the A/A genotype; similarly, increased EGF expression was reported in the serum and liver tissue from HCC patients with the G/G genotype [9].

The aim of this study was to detect the correlation between the EGF gene polymorphism and HCC prediction in Egyptian HCV cirrhotic patients and detection of EGF protein expression in HCC tissue related to this polymorphism.


  Patients and methods Top


This case–control study included 75 patients and 25 apparently healthy individuals matched in age and sex as a control group. Patients were recruited from HCC Clinic, National Liver Institute, Menoufia University, between February 2017 and February 2018. The study was conducted sfter approval of the ethical committee of the faculty and according to the Declaration of Helsinki. All participants provided written informed consent before participation in the study.

All patients provided written consent before study enrollment, and were classified according to clinical examination, laboratory, and imaging findings as follows: group I: 50 cirrhotic HCV patients with HCC, who were subdivided as follows: IA: 25 patients with surgical resectable HCC and IB: 25 patients with advanced unrespectable HCC (multicentric hepatic focal lesions with and/or portal vein thrombosis). Group II included 25 cirrhotic HCV patients with no evidence of HCC. Group III included 25 healthy individuals as the control group. They had normal clinical examination with no history of liver disease, normal laboratory investigations and normal abdominal ultrasonography (US).

Patients with acute and chronic hepatitis, hereditary hepatic diseases, autoimmune liver disorders, other liver cancers, metastatic liver cancer, liver disease other than HCV, and a history of radiological intervention for the management of HCC patients were excluded.

The diagnosis of HCV-related cirrhosis was made by assessment of history, clinical evaluation, laboratory investigations, and abdominal US. HCC was diagnosed by abdominal US, abdominal triphasic computed tomography, and serum alpha fetoprotein level. Detection of EGF in HCC tissue specimens was performed by immunohistochemical examination in patients who underwent liver resection after providing informed consent (subgroup IA).

Laboratory investigations

Ten milliliters of venous blood was withdrawn from all participants included in this study by clean venipuncture from the cubital vein; whole-blood samples were stored at −80°C until assayed. Laboratory investigations and alpha fetoprotein were performed in duplicate according to the manufacturer's instructions.

Epidermal growth factor genotyping

The EGF 61A>G polymorphisms were determined using restriction fragment length polymorphism PCR. Total DNA was extracted using the Zymo Quick-gDNA MiniPrep DNA Purification Kit (Zymo research, Irvine, CA ,United States). EGF genotyping polymorphism was produced for at position 61 (A>G) [single nucleotide polymorphism (SNP) rs4444903] utilizing the 5' nuclease measure TaqMan Assay (TaqMan Universal Master Mix II, Beckman Coulter, California, USA) with allele-specific probes provided by the manufacturer. The following primer sequences were used for PCR amplification of genomic DNA: forward primer sequence: 5'TGTCACTAAAGGAAAGGAGGT-3' and reverse: 5' TTCACAGAGTTTAACAGCCC-3'. PCR amplification using 10 ng of genomic DNA was performed in a thermal cycler (Thermo Fisher Scientific Corp., Waltham, Massachusetts, U.S). Twenty microliters of the reaction mixture was loaded directly and separated by electrophoresis in a 3% agarose gel and stained with ethidium bromide. The gel was visualized on a 302 nm ultraviolet transillumination for genotype detection. The GG genotype yielded 15, 34, and 193 bp fragments. The AA genotype yielded 15, 34, 91, and 102 bp fragments. The AG genotype yielded 15, 34 193, and 102 bp fragments.

Detection of epidermal growth factor in hepatocellular carcinoma tissue of patients who underwent liver resection

Two biopsies were taken from each patient in group IA: one from neoplastic liver tissue and another from adjacent non-neoplastic tissue. Both were stained with hematoxylin and eosin for routine histopathological examination under a light microscope and then stained immunohistochemically for EGF using the streptavidin–biotin amplified system. EGF expression was considered positive when more than or equal to 5% of the cells showed cytoplasmic brown staining.

Statistical analysis

Results were analyzed statistically using the statistical package of social sciences (SPSS 22.0; IBM/SPSS Inc., Chicago, Illinois, USA). Categorical data were presented as number and percentage, whereas quantitative data were expressed as mean and SD. Comparison of continuous data between more than two groups was performed using one-way analysis of variance for parametric data and the Kruskal–Wallis test for nonparametric data with posttests (Turkey and Dunn test, respectively). The χ2 test was used for comparison between categorical data. P value less than 0.05 was considered significant.


  Results Top


This study showed that in the HCC group (group I), there were 41 men and nine women and their ages ranged between 40 and 67 years (mean age, 57.44 ± 7.41 years), in the cirrhotic group (group II) there were 18 men and seven women and, their ages ranged between 45 and 70 years (mean age, 58.12 ± 6.16 years), and in the control group (group III), there were 16 men and nine women and, their ages ranged between 37 and 70 years (mean age, 55.76 ± 8.28 years). There were insignificant differences between all the groups studied in age and sex (P = 0.498 and 0.219, respectively) [Table 1].
Table 1: Statistical analysis of demographical data in the hepatocellular carcinoma, cirrhosis, and control groups

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For the EGF polymorphism, three genotypes, A/A, A/G, and G/G, were detected. The GG genotype and the G allele were more predominant in the HCC group (risk gene), 40 and 63%, respectively, whereas the AA genotype and the A allele were more predominant in the control group (protective gene), 36 and 62%, respectively. AG was less frequent in the HCC group than in the cirrhotic and control groups, which showed statistically significant differences (P = 0.029, 0.005), in the genetic distribution and allele frequencies among the three groups studied [Table 2].
Table 2: Genotype distribution and allele frequencies of the epidermal growth factor polymorphism (61 A/G) in the hepatocellular carcinoma, cirrhosis, and control groups

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The GG genotype was associated with a significantly increased risk of HCC compared with the AA genotype (P = 0.031). However, the AG genotype also had a nonsignificant risk compared with the AA genotype (P = 0.255). The G allele carried a highly significant risk of HCC compared with allele A (P = 0.015). The variant G allele also showed a significant association with HCC risk in the recessive model GG versus AG + AA (P = 0.036) rather than the dominant model GG + AG versus AA (P = 0.66) in comparison between the HCC and the cirrhotic group [Table 3].
Table 3: Comparison of genotype distribution and allele frequencies of the epidermal growth factor polymorphism (61 A/G) in hepatocellular carcinoma versus cirrhotic patients

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EGF expression in tumor tissue (T) was higher in the GG genotype than AG and AA genotypes, and this was statistically different (P = 0.019), whereas in cirrhotic tissue (C), there was no statistical difference (P = 0.626) in the EGF levels among the three genotypes. Comparison between the GG and AG genotypes, in the EGF levels, showed a statistically significant difference (P1 = 0.032), which means that the GG genotype modified the production and the level of EGF in tumor tissue more than the AG genotype [Table 4]. Cancer cells showed cytoplasmic dark brown staining as shown in [Figure 1].
Table 4: Statistical analysis of biochemical and clinical parameters of genotypes of epidermal growth factor polymorphism (61 A/G) in resectable hepatocellular carcinoma patients

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Figure 1: Immunohistochemical staining of resected hepatocellular carcinoma tissues for epidermal growth factor (EGF). Arrows show cancer cells with strong dark brown EGF expression in the cytoplasm and cell membranes. (Immunoperoxidase ×400): immunoperoxidase staining technique with picture magnification 400 times.

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  Discussion Top


Liver carcinogenesis is a complex and multifactorial process, in which both environmental and genetic features interfere and contribute toward malignant transformation [10]. A SNP for a potentially pathogenic gene, such as the EGF gene, might increase HCC prediction improving pretreatment strategies in patients with liver disease [11].

The present study showed that the G allele was the most dominant in HCC patients, 63% of HCC patients and 42% in cirrhotic patients, whereas the A allele was the most prevalent in the control group (62% of controls). The GG and fewer AG genotypes were associated with a significantly increased risk of HCC (5.7 and two folds), respectively, compared with the AA genotype. The G allele carried a highly significant risk of HCC (2.3 folds) compared with allele A.

These results are in agreement with those of Tanabe et al. [12], in two case–control studies, and showed that in patients with hepatitis C-associated cirrhosis, the EGF 61*G allele is highly associated with an increased risk of HCC compared with the A allele; at the same time, patients with the G/G and A/G genotypes had a four and 2.4 fold for HCC risk, respectively, compared with A/A genotype patients. These results proved that the G allele may lead to the risk of hepatocarcinogenesis, whereas the A allele is protective.

Another study was carried out by Abu Dayyeh et al. [13]. In agreement with the present study, in patients with hepatitis C cirrhosis, followed up prospectively for a median time period of 6.1 years to determine and predict the risk of HCC, patients with EGF genotype G/G had a higher risk for HCC than those with genotype A/A.

Zhong et al. [14] carried a meta-analysis on eight studies and reported that an increased risk of HCC was associated significantly with the EGF 61*G allele on the basis of allelic contrast. Patients who carried the EGF 61*A/A genotype had a significantly lower risk of HCC than those with the A/G or the G/G genotype.

In agreement with the present study, a Japanese study was carried out by Suenaga et al. [15]; it was found that there were three genotypes of the EFG gene. The G allele, especially the GG genotype, carried a higher risk for development of HCC than the A/A genotype in Japanese hepatitis C patients.

Abbas et al. [16] reported similar results to our study which showed a significant difference between both patients with HCC and HCV versus controls in terms of the G carrier (GG and GA; 80 vs. 40%, P < 0.05). In addition, GG had three fold odds ratio for developing HCC in the cirrhotic and chronic hepatitis C patients.

Yoshiya et al [17] reported similar results to the present study, but from another prescriptive which showed the GG genotype as the risk genotype for HCC and the AA genotype as the protective one. To investigate the effect of the EFG polymorphism on the recurrence of HCC after curative hepatectomy in patients with chronic HCV infection; it was found that The EGF *61 GG allele was predominant in HCC patients more than AG and AA. The AA genotype had a protective role proved by the lower rate of early recurrence and intra-hepatic metastasis, and lower serum EGF concentration in patients having this genotype compared with patients having the AG or GG genotypes.

Two additional case–control studies were carried out by Yuan et al. [18] among non-Asians in Los Angeles Country, and showed that in patients who had the high-activity 61*G allele of the EGF gene, HCC risk was 78% compared with those had the EGF A/A genotype.

In contrast to the present results, the study carried out by Qi et al. [19] failed to find a significant association between EGF61A/G SNP and the risk of HCC in Chinese patients with chronic HCV infection. The causes for the association between EGF61A/G SNP and the risk of HCC in patients with chronic HCV infection to be controversial among different studies include the different criteria for selection of controls, the small sample size selection in some studies, and the ethnic diversity.

In terms of EGF protein expression in HCC tissue, the present study showed higher concentrations of EGF in the tumor tissue (T) than in the cirrhotic tissue (C), with a significant difference in the GG genotype compared with AG. Li et al. [20] found the same results. The immunohistochemical results of HCC liver tissue showed that samples with the GG genotype expressed EGF protein more than those with the AG genotype. This means that functional polymorphism in the EGF gene can modulate its protein production in the tumor tissue.


  Conclusion Top


To conclude, in the present study, EGF gene polymorphism 61*G was associated with HCC risk and prediction (G/G genotype more than A/G and AA). In addition, increases EGF expression in tumor tissue was associated with the G/G genotype.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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