|Year : 2017 | Volume
| Issue : 4 | Page : 1203-1209
Relationship between mannose-binding lectin-2 gene polymorphism and CD25 with hepatocellular carcinoma-induced hepatitis-C development
Waleed M Abd El Azeem1, Ann A Faried2, Eman A Mahmoud2, Karema A Diab1
1 Department of Clinical Pathology, Faculty of Medicine, National Liver Institute, Menoufia University, Menouf City, Egypt
2 Department of Clinical Pathology; Department of Hepatology, National Liver Institute, Menoufia University, Menouf City, Egypt
|Date of Submission||03-Jan-2017|
|Date of Acceptance||01-Mar-2017|
|Date of Web Publication||04-Apr-2018|
Karema A Diab
Department of Clinical Pathology, National Liver Institute, Menoufia University, Al Hamool, Menouf City, Menoufia Governorate
Source of Support: None, Conflict of Interest: None
The aim of the study was to evaluate the role of mannose-binding lectin-2 ( MBL-2) gene polymorphism and soluble CD25 (sCD25) in the development of hepatitis C-inducing hepatocellular carcinoma (HCC) in Egyptian patients.
Hepatitis C virus (HCV) plays a major role as a cause of chronic liver injury, with potential for neoplastic degeneration. HCC represents an important public health problem in Egypt. MBL is an important constituent of the human innate immune system that acts as an acute-phase reactant and is secreted by the liver. It affects the inflammation severity or disease progression.
Patients and methods
Blood samples from 118 individuals – 88 patients (58 HCC patients and 30 HCV positive patients) and 30 apparently healthy individuals as a control group – were tested for MBL-2 gene polymorphism by real-time PCR and soluble CD25 by using ELISA.
MBL-2 genotype GC was significantly higher among HCC cases than among HCV cases [odds ratio: 8.25 and 95% confidence interval (CI): 2.81–24.24]. Moreover, genotype was significantly more frequent in HCC cases than in HCV cases (odds ratio: 7.22 and 95% CI: 2.67–19.49). On comparing alleles, G allele was of higher rate among HCC cases than among HCV cases (odds ratio: 3.53 and 95% CI: 1.63–7.65). There was a significant increase in (sCD25) level in HCC cases compared with control and HCV groups. CD25 was significantly higher among GG/GC than among CC genotype patients in the HCC group only. In addition, there was significant positive correlation between CD25 and aspartate aminotransferase, total protein, albumin, direct bilirubin, total bilirubin, and α-fetoprotein.
Functionally relevant MBL-2 promoter polymorphism may play a role in the development of HCV-related HCC, and sCD25 can be used to distinguish HCC from appropriate controls with early tumors.
Keywords: CD25, enzyme-linked immmunosorbent assay, gene polymorphism, hepatocellular carcinoma, MBL-2
|How to cite this article:|
Abd El Azeem WM, Faried AA, Mahmoud EA, Diab KA. Relationship between mannose-binding lectin-2 gene polymorphism and CD25 with hepatocellular carcinoma-induced hepatitis-C development. Menoufia Med J 2017;30:1203-9
|How to cite this URL:|
Abd El Azeem WM, Faried AA, Mahmoud EA, Diab KA. Relationship between mannose-binding lectin-2 gene polymorphism and CD25 with hepatocellular carcinoma-induced hepatitis-C development. Menoufia Med J [serial online] 2017 [cited 2020 Apr 6];30:1203-9. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1203/229205
| Introduction|| |
Hepatitis C virus (HCV) plays a major role as a cause of chronic liver injury, with potential for neoplastic degeneration. On a global scale, HCV infection leads to about 350 000 deaths yearly.
Hepatitis C and alcohol are the most widespread causes of liver disease worldwide. Hepatocytes are the main sites of HCV infection, which generate oxidative stress. Oxidative stress levels affect HCV replication and innate immunity, resulting in a greater susceptibility for HCV infection.
Hepatocellular carcinoma (HCC) – a major pathological type of primary liver cancer – is an increasingly prevalent clinical problem worldwide.
Liver cancer is strongly linked to hepatitis B virus (HBV) and HCV. Egypt has the highest prevalence of HCV worldwide (13.8%) and has rising rates of HCC; hospital-based studies in Egypt have reported an increase in the relative frequency of all liver-related cancers in Egypt (95% as HCC), from 4% in 1993 to 7.3% in 2003.
HCC represents an important public health problem in Egypt. In many Egyptian regional registries, liver cancer is the first most common cancer in men and the second in women.
Mannose-binding lectin (MBL) is an important constituent of the human innate immune system, which acts as an acute-phase reactant and is secreted by the liver. It is a calcium-dependent C-type lectin with a structural analogy of component C1q. It can bind through multiple lectin domains to the carbohydrate moieties expressed on the surface of many microbial organisms, and activate macrophages and the complement system cascade. It plays an important role in regulating the production of proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1β produced by monocytes in response to microbial infection and hence may affect the inflammation severity or disease progression.
α-Fetoprotein (AFP), together with hepatic ultrasonography, is the most common marker used in clinical practice to detect HCC, but the clinical value of AFP is challenged in recent years due to its low sensitivity and specificity.
This substandard sensitivity necessitates the need for a biomarker that has the ability to detect HCC at an early stage. IL-2 is an important cytokine in vivo that is able to induce T-cell proliferation and activation after binding to its IL-2 receptor (IL-2R) and then enhance the immune response in vivo. sCD25 is the free form of IL-2Rα subunit, that can competitively bind to IL-2 with IL-2R, and inhibit the proliferation of lymphocyte and downregulate the activity of NK cells, lowering the immune function. Therefore, circulating sCD25 level is considered as an indicator of the degree of immune inhibition. Bien and Balcaerska suggested that in most patients with haematological malignancies, tumor cells continuously produce a large number of sCD25, which is the main reason for the upregulation of. sCD25 is significantly elevated in a small series of HCC patients compared with controls.
This study aimed to evaluate the role of mannose-binding lectin-2 (MBL-2) gene polymorphism and soluble CD25 (sCD25) in the development of hepatitis C-inducing HCC in Egyptian patients.
| Patients and Methods|| |
This study was carried out under ethical contributions at Clinical Pathology Department, National Liver Institute, Faculty of Medicine, Menoufia university, between October 2013 and March 2016. The study was conducted on three groups: the HCC group, which included 58 patients; the HCV group, consisting of 30 patients; and the control group, which included age and sex matched 30 apparently healthy individuals.
Following were the exclusion criteria: antiviral or immunomodulatory therapy, history of excessive alcohol consumption, treatment with hepatotoxic drugs, and HBV infection.
After obtaining an informed consent, all patients and controls included in this study were subjected to a thorough history taking, complete clinical examinations, abdominal ultrasonography (using real-time ultrasound equipment), and laboratory investigations.
Ten ml of blood in plain tube was withdrawn from the patients and controls under complete aseptic condition and left to clot, and then centrifuged. Clear sera were separated and divided into four aliquots; the first one was used to determine liver function tests, the second one was tested for HCV-RNA and HBV-DNA assay quantitatively, the third one was used for measurement of AFP, and the fourth one for CD25. The specimens were kept frozen at −80°C until the time of assay. In addition, 2 ml was collected in vacutainer tubes containing EDTA for DNA extraction.
Liver function tests [serum bilirubin (total and direct), serum aspartate aminotransferase, serum alanine aminotransferase, serum alkaline phosphatase, serum albumin] were supplied by Rochintegra 400 plus; Switzerland (Roche Instrument Ctr Ag Tegimenta, Forrenstrasse, Rotkreuz 6343, Switzerland 6343).
AFP was supplied by Elecsys E411, Switzerland, sCD25 by solid phase sandwitch Quantikine E LISA (Minneapolis, MN 55413, USA) kit, and blood level of MBL-2 gene polymorphism by means of real-time PCR, which is based on these processes:
- DNA extraction: DNA was extracted from 0.5 ml up to 1.0 ml of whole blood using the Gene Jet genomic DNA purification kit (DNA purification columns preassembled with collection tubes) provided by Thermo Fisher Scientific, Waltham, Massachusetts, USA
- PCR was done for the amplification of target DNA using MBL-2-specific complimentary primers (MBL-2 forward: 5′-GCA CGG TCC CAT TTG TTC TCA-3′; MBL-2 reverse: 5′-GCG TTG CTG CTG GGA GAC TAT AAA-3′) and hybridization of the amplified products to fluorogenic probe using Applied biosystem 7500 supplied by the Roche company (Rotkreuz, Switzerland). The amplification of the target was done through the following steps: denaturation (generally, a 2-min initial denaturation step at 95°C is sufficient), annealing (the annealing step is typically 1 min at 65°C), extension (the extension reaction is typically performed at the optimal temperature for the DNA polymerase, which is 72°C), and a final extension of 2 min at 72°C is recommended.
Data were collected, tabulated, and statistically analyzed by using SPSS, version 20 (SPSS Inc., Chicago, Illinois, USA). Sensitivity, specificity, significance of results (P value), odds ratio, and the c2-test were calculated to compare MBL-2 gene polymorphism between HCC, HCV, and control groups to assess MBL-2 gene polymorphism with the development of hepatitis C-inducing HCC in Egyptian patients. Receiver operator characteristic curves for CD25 with respective points of maximal accuracy for sensitivity and specificity were generated to determine biomarker performance. Spearman's rank correlation coefficient was used to examine the correlation between the level of sCD25 and other laboratory parameters.
| Results|| |
[Table 1] illustrates that GC genotype was significantly of higher rate among HCC cases than among controls [odds ratio: 18 and 95% confidence interval (CI): 4.73–68.53]; in addition, GG/CG genotype was significantly more frequent in HCC cases than in controls (odds ratio: 10.5 and 95% CI: 3.62–30.43). On comparing alleles, G allele was of higher rate among HCC cases than among controls (odds ratio: 4 and 95% CI: 1.80–8.89).
|Table 1: Comparison and odds ratio of MBL-2 genotypes between the hepatocellular carcinoma group and the control group|
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[Table 2] demonstrates that the distribution of both MBL-2 genotypes was nonsignificantly different in HCV cases and controls; in addition, GG/CG genotype showed nonsignificant difference in HCV cases and control (odds ratio: 10.5 and 95% CI: 3.62–30.43). On comparing alleles, there was nonsignificant difference regarding alleles in HCV cases and controls.
|Table 2: Comparison and odds ratio of MBL-2 genotypes between the hepatitis C virus group and the control group|
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[Table 3] shows that GC genotype was significantly of higher rate among HCC cases than among HCV cases (odds ratio: 8.25 and 95% CI: 2.81–24.24); in addition, GG/CG genotype was significantly more frequent in HCC cases than in HCV cases (odds ratio: 7.22 and 95% CI: 2.67–19.49). On comparing alleles, G allele was of higher rate among HCC cases than among HCV cases (odds ratio: 3.53 and 95% CI: 1.63–7.65).
|Table 3: Comparison and odds ratio of MBL-2 genotypes between the hepatocellular carcinoma group and the hepatitis C virus group|
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[Table 4] shows a significant statistical difference between the HCC group and the control group as regards AFP and CD25, with higher AFP and higher CD25 in the HCC group compared with the control group. There is a significant statistical difference between the HCV group and the control group as regards AFP and CD25, with higher AFP and higher CD25 in the HCV group compared with the control group. Moreover, there is a significant statistical difference between the HCC group and the HCV group as regards AFP and CD25, with higher AFP and higher CD25 in the HCC group than in the HCV group.
|Table 4: Comparison between cases and control groups as regards α-fetoprotein and CD25|
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[Table 5] shows that AFP was significantly higher among GG/GC than among CC genotype patients in both HCC and HCV groups, and CD25 was significantly higher among GG/GC than among CC genotype patients in the HCC group only.
[Table 6] shows significant positive correlation between CD25 and aspartate aminotransferase, total protein, albumin, direct bilirubin, total bilirubin, and AFP, whereas there is nonsignificant correlation between it and γ-glutamyl transferase, alkaline phosphatase, viral load, child score, and tumor size.
[Table 7] shows validity of CD25 for prediction of HCC cases from HCV cases, with an area under the curve of 0.96 for CD25 and 0.93 for AFP. At cutoff point 5830 pg/ml for CD25, its sensitivity was 84.5%, specificity was 96.7%, negative predictive value was 76.3%, positive predictive value was 98%, and accuracy was 88.6%. Whereas at cutoff point 12.7 ng/ml for AFP, its sensitivity was 82.8%, specificity was 73.3%, negative predictive value was 68.8%, positive pre dictive value was 85.7%, and accuracy was 79.5% required [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6].
|Table 7: Validity of CD25 and α-fetoprotein for prediction of hepatocellular carcinoma cases from hepatitis C virus cases|
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|Figure 1: Relationship between MBL-2 genotypes and CD25 in the hepatocellular carcinoma (HCC) group.|
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|Figure 2: α-Fetoprotein of the studied groups. HCC, hepatocellular carcinoma; HCV, hepatitis C virus.|
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|Figure 3: CD25 of the studied groups. HCC, hepatocellular carcinoma; HCV, hepatitis C virus.|
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|Figure 4: Positive correlation between CD25 and α-fetoprotein. HCC, hepatocellular carcinoma.|
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|Figure 5: Receiver operating characteristics (ROC) curve of CD25 to predict hepatocellular carcinoma cases from hepatitis C virus cases.|
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|Figure 6: Receiver operating characteristics (ROC) curve of a-fetoprotein to differentiate between hepatocellular carcinoma cases and hepatitis C virus cases.|
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| Discussion|| |
HCC comprises nearly 6% of all annual incident cancer cases worldwide and accounts for 85–90% of all primary liver tumors. HCC is an increasingly prevalent clinical problem worldwide. MBL-2 plays an important role in the innate immune system, acting as an opsonin factor by activation of antibody-independent pathway of the complement system. Impaired production of MBL-2 may therefore cause insufficient complement activation, thus facilitating disease susceptibility and progression and a significantly increased risk for the development of severe infections. Circulating sCD25 level is considered as an indicator of the degree of immune inhibition. Ehsan et al. suggested that in most patients with hematological malignancies, tumor cells continuously produce a large number of sCD25, which is the main reason for the upregulation of sCD25. The present study was planned to evaluate the role of MBL-2 gene polymorphism and sCD25 in the development of hepatitis C-inducing HCC in Egyptian patients.
The present study found that GC genotype was significantly of higher rate among HCC cases than among controls (odds ratio: 18 and 95% CI: 4.73–68.53); in addition, GG/CG genotype was significantly more frequent in HCC cases than in controls (odds ratio: 10.5 and 95% CI: 3.62–30.43). On comparing alleles, G allele was of higher rate among HCC cases than among controls (odds ratio: 4 and 95% CI: 1.80–8.89). Moreover, GG/CG genotype showed nonsignificant difference in HCV cases and controls (odds ratio: 10.5 and 95% CI: 3.62–30.43). On comparing alleles, there was nonsignificant difference regarding alleles in HCV cases and controls, whereas GC genotype was significantly of higher rate among HCC cases than among HCV cases (odds ratio: 8.25 and 95% CI: 2.81–24.24); in addition, GG/CG genotype was significantly more frequent in HCC cases than in HCV cases (odds ratio: 7.22 and 95% CI: 2.67–19.49). On comparing alleles, G allele was of higher rate among HCC cases than among HCV cases (odds ratio: 3.53 and 95% CI: 1.63–7.65). Our results were in line with those of a study by Halla et al. conducted on 232 definitely HCV-negative patients, suggesting higher susceptibility for HCV infection when G allele is present. In contrast, Baccarelli et al. demonstrated lower C allele frequency in 412 randomly selected European controls with unknown HCV status (G: 24.0, C: 76.0%). These apparently controversial findings are not necessarily surprising. Originating from substantially different populations, those that were not exposed to the same risk of HCV infection and HCC development should be regarded at least with caution or even as incomparable. Segat et al. had previously carried out a similar study on the role of other MBL-2 gene polymorphisms (exon-1) in the development of virus-related HCC (HBV and HCV) in a cohort with comparable sample size (n = 99) using healthy controls who were not exposed to same HCV-related risk as study population. They could not demonstrate any association between MBL-2 genotypes and prevalence of HCC. Bataller and Brenner showed that the presence of C allele might indeed result in lower HCV susceptibility, lower incidence of further HCV-related complications, and their extent, as long as study population remains homogenous (e.g. race, age, and sex) and is exposed to the same risk of disease development. Similarly, Rantala et al. concluded the potential ability of C allele to induce higher levels of MBL, thus affecting HCV susceptibility and disease progression. In the present study, there were statistically higher percent of positive AFP in patients with genotyping GG/GC than in patients with negative AFP. Neumann et al. showed that the association of MBL-2 polymorphisms with HCC occurrence, size, growth pattern, and AFP levels support the theory of genetic impact on disease development. Dennis et al. found significantly higher levels of AFP in HCC patients with MBL-2G allele. This genetic variation may contribute to the pathogenesis of HCC, and similarly to the whole variety of already discovered genetic risk factors for carcinogenesis. The significant association of apparently protective CC genotype with AFP-negative HCCs might serve as a supplementary diagnostic tool. Cervera et al. reported that MBL-2G allele was found to be associated with the presence and size of HCC, as well as with bilobar tumor growth and AFP levels. We found that validity of CD25 for prediction of HCC cases from HCV cases with cutoff point 5830 pg/ml, sensitivity was 84.5% and specificity was 96.7%. Ehsan et al. reported that sCD25 levels were significantly increased in HCC versus control and cirrhotic groups and by using a cutoff value of 1425 pg/ml; sCD25 had a sensitivity of 64% and specificity of 96.15%.
In the current study there was no significant difference between CD25 levels and tumor size, and this was in agreement with Durazo et al. who found no significant difference between patients according to their tumor size (P< 0.1416); however, the AFP level in serum increased with size.
| Conclusion|| |
On the basis of the previous finding, it could be concluded that the functionally relevant MBL-2 promoter polymorphism may play a role in the development of HCV-related HCC. Further investigations are needed in larger prospective trials including other disease entities combined with other candidate gene variants involved in carcinogenesis. Moreover, an evaluation of both serum levels of AFP and CD25 as markers for HCC is.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zaltron S, Spinetti A, Biasi L, Baiguera C, Castelli F. Chronic HCV infection: epidemiological and clinical relevance. BMC Infect Dis 2012; 12(Suppl 2)
Osna N, Ganesan M, Kharbanda K. Hepatitis C, innate immunity and alcohol friends or foes?. Biomolecules 2015; 5
Zhang B, Zhang X, Zhou T, Liu J. Clinical observation of liver cancer patients treated with axitinib and cabozantinib after failed sorafenib treatment. Cancer Biol Ther 2015; 26
Lehan E, Wilson M. Epidemiology of hepatitis viruses among hepatocellular carcinoma cases and healthy people in Egypt: a systematic review and meta-analysis. Int J Cancer 2009; 124
Baghdady I, Fouad F, Sayed M, Shoaib A, Salah Y, Elshayeb E, et al.
Serum markers for the early detection of hepatocellular carcinoma in patients with chronic viral hepatitis C infection. Menoufia Med J 2014; 27
Hang-di X, Ming-fei Z, Tian-hong W, Guang-zhong S. Association between mannose-binding lectin gene polymorphisms and hepatitis B virus infection: a meta-analysis. Plos One 2013; 35
Mariam A, Emad F, Waleed F, Dalia H, Osama H. Golgi protein 73 versus alpha fetoprotein as a marker for hepatocellular carcinoma. Menoufia Med J 2016; 29
Bien E, Balcerska A. Serum soluble interleukin 2 receptor alpha in human cancer of adults and children: a review. Biomarkers 2008; 13
Cabrera R, Fitian AI, Ararat M, Xu Y, Brusko T, Wasserfall C, et al.
Serum levels of soluble CD25 as a marker for hepatocellular carcinoma. Oncol Lett 2012; 4
Hu SR, Liu JM, Yang TL, Liu HZ, Huang JL, Lin QW, et al.
Determination of human alpha-fetoprotein (AFP) by solid substrate room temperature phosphorescence enzyme-linked immune response using luminescent nanoparticles. Microchim Acta 2005; 152
Ng WF, Duggan PJ, Ponchel F, Matarese G, Lombardi G, Edwards AD, et al.
Human CD4+CD25+cells: a naturally occurring population of regulatory T cells. Blood 2001; 98
Martin C, Chen S, Maya-Mendoza A, Lovric J, Sims PF, Jackson DA. Lamin B1 maintains the functional plasticity of nucleoli. J Cell Sci 2009; 122
El-Serag H, Davila J. Surveillance for hepatocellular carcinoma in whom and how. Ther Adv Gastroenterol. 2011; 4
Venook A, Papandreou C, Furuse J, de Guevara L. The incidence and epidemiology of hepatocellular carcinoma: a global and regional perspective. Oncologist 2010; 15(Suppl 4)
Eurich D, Neumann UP, Boas-Knoop S, Neuhaus R, Bahra M, Neuhaus P, et al.
Role of mannose binding lectin polymorphism in the development of acute cellular rejection after transplantation for hepatitis C virus-induced liver disease. Transpl Infect Dis 2012; 14
Ehsan R, Sahar Z, Ahmed A, Abbas N, et al
. Soluble CD25 and hepatocellular carcinoma in. Int J Adv Res 2015;5:658–664
Halla M, Carmo R, Silva L. Association of hepatitis C virus infection and liver fibrosis severity with the variants alleles of MBL2 gene in a Brazilian population Hum Immunol 2010; 71
Baccarelli A, Hou L, Chen J. Mannose-binding lectin-2 genetic variation and stomach cancer risk. Int J Cancer 2006; 119
Segat L, Fabris A, Padovan L. MBL2 and MASP2 gene polymorphisms in patients with hepatocellular carcinoma. J Viral Hepat 2008; 15
Bataller R, Brenner D. Liver fibrosis. J Clin Invest 2005; 115
Rantala A, Lajunen T, Juvonen R, Bloigu A, Silvennoinen-Kassinen S, Peitso A, et al.
Mannose-binding lectin concentrations, MBL2 polymorphisms, and susceptibility to respiratory tract infections in young men. J Infect Dis 2008; 198
Neumann U, Puhl G, Bahra M, Berg T, Langrehr JM, Neuhaus R, et al.
Treatment of patients with recurrent hepatitis C after liver transplantation with peginterferon alfa-2B plus ribavirin. Transplantation 2006; 82
Eurich D, Boas-Knoop S, Morawietz L, Neuhaus R, Somasundaram R, Ruehl M, et al
. Association of mannose-binding lectin-2 gene polymorphism with the development of hepatitis C-induced hepatocellular carcinoma. Liver Int 2011; 31
:1006–1012. doi: 10.1111/j.1478-3231.2011.02522.x.[PubMed] [Cross Ref]
Cervera C, Balderramo D, Suarez B, et al
. Donor mannose binding lectin gene polymorphisms influence the outcome of liver transplantation. Liver Transpl 2009; 15
Durazo FA, Blatt LM, Corey WG, Lin JH, Han S, Saab S, Busuttil RW, Tong MJ. Des-gamma-carboxyprothrombin, alpha-fetoprotein and AFP-L3 in patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma. J Gastroenterol Hepatol 2008; 23
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]