|Year : 2016 | Volume
| Issue : 4 | Page : 783-788
Soluble fas levels as a marker in chronic hepatitis C
Ahmed B Mahmoud1, Enas M Ghoneim2, Azza M Abd El-Aziz2, Shimaa M Lotfy Abass MBBCH 3
1 Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 National Liver Institute, Menoufia University, Menoufia, Egypt
3 El-Bagour Hospital, Shebin Elkom, Egypt
|Date of Submission||21-May-2014|
|Date of Acceptance||02-Sep-2014|
|Date of Web Publication||21-Mar-2017|
Shimaa M Lotfy Abass
Department of Medical Microbiology and Immunology, Faulty of Medicine, Menoufia University, El gish Street, El Bagor, Menoufia Governorate
Source of Support: None, Conflict of Interest: None
The aim of the study was to investigate the effect of soluble Fas (sFas) on patients with chronic hepatitis C.
Programmed cell death has been observed in leukocytes among patients with chronic Hepatitis C and hepatocellular carcinoma (HCC), and now there is a strong trend to use sFas as a predictive marker for chronic hepatitis C and tumorigenesis in HCC.
Seventy patients were divided into three groups. Group I included 30 patients with chronic hepatitis C infection due to hepatitis C virus (HCV) (cirrhotic and noncirrhotic). Group II included 20 patients with HCC with HCV infection. Group III included 20 healthy individuals without hepatitis C infection or any other disease, which served as the control group. Liver function tests, complete blood count, analysis of viral markers, analysis of HCV-RNA by PCR, and evaluation of serum sFas by enzyme-linked immunosorbent assay were carried out.
Serum sFas level increases in chronic hepatitis C and HCC, with positive correlation between sFas and aspartate aminotransferase, alanine aminotransferase, total bilirubin, and direct bilirubin and negative correlation between sFas and Hb level, platelet count, serum albumin, prothrombin time, and viral load (PCR). sFas at cutoff of 6581.12 could predict patients with HCV with 83.3% sensitivity and 65% specificity. sFas at cutoff of 6960.91 could predict patients with HCC with 85% sensitivity and 30% specificity. sFas at cutoff of 9125.99 could discriminate between cirrhotic and noncirrhotic HCV patients with 80.0% sensitivity and 53.3% specificity.
sFas can be considered a predictive marker for chronic hepatitis C and tumorigenesis in HCC.
Keywords: chronic hepatitis C, hepatocellular carcinoma, soluble Fas
|How to cite this article:|
Mahmoud AB, Ghoneim EM, Abd El-Aziz AM, Lotfy Abass SM. Soluble fas levels as a marker in chronic hepatitis C. Menoufia Med J 2016;29:783-8
|How to cite this URL:|
Mahmoud AB, Ghoneim EM, Abd El-Aziz AM, Lotfy Abass SM. Soluble fas levels as a marker in chronic hepatitis C. Menoufia Med J [serial online] 2016 [cited 2017 Dec 18];29:783-8. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/783/202499
| Introduction|| |
Hepatitis C virus (HCV) infection is a frequent cause of acute and chronic hepatitis worldwide and creates a significant burden on healthcare systems because of the high morbidity and mortality .
The majority of chronic cases of HCV are associated with liver inflammation and slow progression of hepatic fibrosis. This progressive course leads to cirrhosis in as many as 20% of patients. It also has been estimated that ˜1–4% of patients with cirrhosis will develop hepatocellular carcinoma (HCC) each year .
Apoptosis (programmed cell death) is an active form of cell death that is initiated by a number of stimuli. Apoptosis is often associated with characteristic morphological and biochemical features, including changes in the plasma membrane, cell shrinkage, chromatin condensation, and chromosomal fragmentation .
The mechanisms of apoptosis are highly complex and involve an energy-dependent cascade of molecular events: the death receptor pathway, the mitochondrial pathway, and the perforin/granzyme pathway .
Fas/Apo-1 (CD95) is a type1 transmembrane glycoprotein that signals sensitive cells to die by apoptosis upon ligation with the natural ligand FasL, activating caspase-8 and eventually caspase-3, (both members of the tumor necrosis factor superfamily), which ends in cell death by apoptosis .
Fas receptors are widely expressed in normal and diseased tissues. It has been implicated in tumor progression of several cancers. FasL is expressed mainly in cytotoxic T lymphocytes, in immune privileged sites, and in various tumors in which specific cytotoxic T-cell clones are produced .
Apoptosis is tightly regulated through a variety of mechanisms, one of which is postulated to be the production of soluble Fas (sFas), an antagonistic decay protein similar to Fas, exceptionally lacking the transmembrane domain. It normally binds to FasL and blocks the signaling of the membrane-bound form of Fas. Elaboration of sFas by tumor cells by alternative mRNA splicing may contribute to resistance to Fas-mediated apoptosis .
Elevation of the level of serum sFas in chronic hepatitis C and HCC has been observed by El Bassiouny et al. .
The present work aimed to characterize sFas in patients with chronic hepatitis C and investigate whether it has a role in disease pathogenesis.
| Methods|| |
The study population included 50 patients selected from clinics of the National Liver Institute, Menoufia University, and Shebin El-Kom Teaching Hospital and were divided into two groups. Group I included 30 patients with chronic hepatitis C due to HCV infection, which was subdivided into the cirrhotic group (15 patients, nine male and six female; aged 23–59 years) and the noncirrhotic group (15 patients, 11 male and four female; aged 26–59 years). Group II included 20 patients with HCC with HCV infection (15 male and five female; aged 18–59 years). Twenty healthy individuals without any disease served as the control group (16 male and four female; aged 27–54 years).
All patients and controls were matched for age and sex. Informed consent was obtained from every patient and control in accordance with the local ethical committee. All individuals were subjected to complete history taking, complete clinical examination, and abdominal ultrasonography.
Blood samples from patients and controls were allowed to clot naturally in test tubes; serum was then separated by centrifugation, divided into small aliquotes, and stored immediately at −20°C until use for liver function tests and analysis of HCV-Ab, HBsAg by enzyme-linked immunosorbent assay, HCV-RNA by PCR, and serum sFas by enzyme-linked immunosorbent assay [9–12].
Serum samples required at least a 10-fold dilution with Calibrator Diluent RD5L (1×). sFas standard was reconstituted with 1.0 ml of deionized or distilled water. The 2000 pg/ml standard served as the high standard (Eli Lilly and Company, Indianapolis, USA). Calibrator Diluent RD5L (1×) served as the zero standard (0 pg/ml). A volume of 100 µl of Assay Diluent RD1-8 was added to each well; 100 µl of standard, control, or sample was also added per well, and the wells were incubated for 2 h at room temperature. Each well was washed and aspirated five times by diluted wash buffer solution. sFas conjugate measuring 200 µl was added to each well and the wells were incubated for 2 h at room temperature. The aspiration/wash was repeated. Substrate solution of 200 µl was added to each well and incubation was carried out for 30 min at room temperature. Thereafter, 50 µl of stop solution was added. The color in the wells should change from blue to yellow. The optical density of each well was measured within 30 min, using a microplate reader set to 450 nm and correction at 540 nm.
Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best-fit curve through the points on a log/log graph. The data may be linearized by plotting the log of the sFas concentrations versus the log of the OD on a linear scale, and the best-fit line can be determined by regression analysis. As the samples were diluted 10-fold, the concentration read from the standard curve must be multiplied by10.
The t-test is used to assess the statistical significance of difference between two means. By knowing the t-test and the degree of freedom, the P-value is calculated from special tables, and thus the significance of the results was determined from the 't' distribution tables.
P > 0.05 = insignificant difference; P< 0.05 = significant difference.
P< 0.01 = highly significant difference; P< 0.001 = very highly significant difference.
Sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy were calculated. The predictive values of the studied parameters were compared with the control group data by means of the receiver operating characteristic curve. The data were expressed as area under the curve. All reported P-values are based on two-sided tests and compared with the patient group significance level of 5% .
| Results|| |
In this study, there were 15 male (75%) and five female (25%) patients in the HCC group, their ages ranging between 18 and 59 years with a mean of 38.30 ± 12.42 years. There were 11 male (73.3%) and four female (26.7%) patients in the noncirrhotic group, their ages ranging between 26 and 59 years with a mean of 38.80 ± 9.7 years. There were nine male (60%) and six female (40%) patients in the cirrhotic group, their ages ranging between 23 and 57 years, with a mean of 37.87 ± 10.78 years, and 16 male (80%) and four female (20%) patients in the control group, their ages ranging between 27 and 54 years, with a mean of 39.05 ± 7.99 years. There was no statistically significant difference among the groups ([Table 1]).
There was no statistically significant difference in the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), direct bilirubin, and prothrombin time (PT) between HCC and HCV patients; however, total bilirubin was significantly higher in HCC patients in comparison with HCV patients. There was a statistically significant difference in all liver functions between HCC patients and the control group and between HCV patients and the control group ([Table 2]).
|Table 2 The mean values of laboratory parameters in the three studied groups|
Click here to view
Hemoglobin level and platelet counts were significantly lower among HCC cases than among HCV cases and controls, and also significantly lower among HCV patients compared with controls. There was no significant difference in white blood cell count between the three groups ([Table 3]).
We found no statistically significant difference in viral load level between cirrhotic and noncirrhotic HCV patients, nor between HCV and HCC patients ([Table 4]).
sFas was statistically significantly higher in HCC and HCV patients in comparison with controls. However, there was no significant difference between HCC and HCV patients [Figure 1].
There was significant positive correlation between sFas and AST, ALT, total bilirubin, and direct bilirubin, and significant negative correlation between sFas and Hb level, platelet count, serum albumin, prothrombin time, and viral load ([Table 5]).
|Table 5 Correlation between soluble Fas and other parameters in the patient groups|
Click here to view
sFas at cutoff of 6581.12 could predict patients with HCV with 83.3% sensitivity and 65% specificity [Figure 2].
|Figure 2: Receiver operating characteristic (ROC) curve of soluble Fas differentiating patients with hepatitis C virus (HCV).|
Click here to view
sFas at cutoff of 6960.91 could predict patients with HCC with 85% sensitivity and 30% specificity [Figure 3].
|Figure 3: Receiver operating characteristic (ROC) curve of soluble Fas level differentiating patients with hepatocellular carcinoma (HCC).|
Click here to view
sFas at cutoff of 9125.99 could discriminate between cirrhotic and noncirrhotic patients in the HCV group with 80.0% sensitivity and 53.3% specificity [Figure 4].
|Figure 4: Receiver operating characteristic (ROC) curve of soluble Fas for differentiating between cirrhotic and noncirrhotic patients in the hepatitis C virus (HCV) group.|
Click here to view
| Discussion|| |
The present work aimed to characterize sFas in patients with chronic hepatitis C and to investigate whether it has a role in disease pathogenesis.
Our study included 15 male (75%) and five female (25%) patients with HCC, their ages ranging between 18 and 59 years, with a mean of 38.30 ± 12.42 years. There were 11 male (73.3%) and four female (26.7%) patients in the noncirrhotic group, their ages ranging between 26 and 59 years, with a mean of 38.80 ± 9.7 years. There were nine male (60%) and six female (40%) patients in the cirrhotic group, their age ranging between 23 and 57 years, with a mean of 37.87 ± 10.78 years, and 16 male (80%) and four female (20%) patients in the control group, their ages ranging between 27 and 54 years, with a mean of 39.05 ± 7.99 years. There was no statistically significant difference between the groups (P > 0.05). A comparable mean age of 39.14 ± 12.76 years in patients with HCV infection was reported by Mohamed et al.  Male predominance in HCV-infected patients was also reported by Feier et al. .
This study showed statistically significant elevation in ALT and AST and significant lowering of albumin and prothrombin in HCC and HCV patients compared with the control group. This is because ALT and AST indicate liver cell damage, whereas albumin and prothrombin are indicators of synthetic functions of the liver. In another study, there was no statistically significant difference in liver function between HCC and HCV patients .
In this study there was no significant difference in white blood cell count between the three studied groups, whereas hemoglobin level was significantly lower in HCV patients than in controls and also in HCC patients compared with HCV patients and controls. Similarly, another study reported that HCC and HCV patients develop anemia . They attributed this to two reasons:First, ribavirin, one of the drugs used to treat hepatitis C, often causes anemia, which is usually mild. Second, the presence of cirrhosis makes the spleen eliminate too many red blood cells from circulation or decreases the production of red blood cells.
Also, platelet count was significantly lower among HCV patients than among controls and among HCC cases than among HCV cases and controls in another study, which reported that thrombocytopenia is a common finding in patients with chronic hepatitis C. The etiology of this thrombocytopenia is still obscure. There is increasing interest in the potential role of thrombopoietin as a cause of this thrombocytopenia .
In the current study, no significant difference was found among HCV cases and HCC cases with respect to viral load level. Nawaz et al.  reported the same result. They reported that viral load did not provide information on the stage of disease because there was little or no correlation between the HCV viral load and the extent of hepatic fibrosis or risk of disease progression but it had prognostic value with regard to response to antiviral therapy.
The virus tries to inhibit apoptosis to prolong the life of the cell and thereby maximize the number of progeny virion. In contrast, the host should stimulate apoptosis, thereby inhibiting viral growth and blocking the viral spread .
This study showed that the level of serum sFas in chronic hepatitis C was significantly higher than that in healthy individuals. Similarly, another study reported increased levels of sFas in the serum of patients with HCV in comparison with controls [20–22]. They attributed this to increased number of Fas-positive cells and decreased clearance of sFas from the liver.
This study also showed that sFas level was significantly higher among patients with HCC compared with the control group. Other studies reported the same result ,. This could be explained by the role of sFas in the inhibition of apoptosis by binding to FasL and blocking the signaling of the membrane-bound form of Fas. This means that sFas may function as an inhibitor of the Fas/FasL system and thus the tumor cells can escape from immune surveillance.
We found that there was no significant difference in serum sFas concentration between HCC and HCV patients. Similarly, Lee et al.  reported that apoptosis and the Fas system were significantly involved in the process of converting liver cirrhosis into HCC.
Roskams et al.  reported that sFas was significantly lower in HCC than in HCV samples. This was attributed to the fact that tumor cells possess more than one safeguard against Fas-mediated apoptosis. They explained the inhibition of apoptosis as follows: first, the reduced expression or loss of certain molecules that are involved in the Fas-mediated apoptosis pathway, such as FADD (Fas-associated protein with death domain) or FAF (Fas-associated factor); second, the induction of molecules that would inhibit Fas-mediated apoptosis, such as FAP (Fas-associated phosphatase); and third, Fas mutation could be expected.
This study showed that there was significant positive correlation between sFas and AST, ALT, and bilirubin. Similarly, another study showed that there was positive correlation between sFas and liver functions .
We found that there was significant negative correlation between sFas and hemoglobin level. A high level of sFas may play an important pathogenetic role in dyserythropoesis, as reported in a previous study .
Our results showed that sFas at cutoff of 6581.12 pg/ml could predict patients with HCV with 83.3% sensitivity and 65% specificity. Nearly similar results were reported by Kern et al. .
We found that sFas at cutoff of 6960.91 pg/ml could predict patients with HCC with 85% sensitivity and 30% specificity. Nearly similar results were reported by Valva et al. .
We also found that sFas at cutoff of 9125.99 pg/ml could discriminate between cirrhotic and noncirrhotic patients in the HCV group with 80.0% sensitivity and 53.3% specificity. A nearly similar result was reported by Valva et al. , who reported that sFas at cutoff of 13 806.67 pg/ml could discriminate between cirrhotic and noncirrhotic HCV patients with 100% sensitivity and 70.6% specificity.
| Conclusion|| |
Serum sFas level increases in chronic hepatitis C and HCC patients with positive correlation between sFas and AST, ALT, total bilirubin, and direct bilirubin and negative correlation between sFas and Hb level, platelet count, serum albumin, prothrombin time, and viral load (PCR). sFas could be considered a predictive marker for chronic hepatitis C and tumorigenesis in HCC.
| Acknowledgements|| |
Conflicts of interest
| References|| |
Schiavon LL, Narciso JL, Carvalho RJ. Evidence of a significant role for Fas-mediated apoptosis in HCV clearance during pegylated interferon plus ribavirin combination therapy. Antiviral Ther 2011; 16
Wagida AK, Anwar A, Hussein M Khaled B. Changing pattern of hepatocellular carcinoma (HCC) and its risk factors in Egypt: possibilities for prevention, Mutat Res 2008; 659
Lee-Anne F. Small molecular weight modulators of apoptosis. Am Clin Biochem 2011; 14
Dwyer DJ, Camacho DM, Kohanski M. Antibiotic-induced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis. Mol Cell 2012; 46
Rastogi R, Sinha RP. Review article apoptosis: molecular mechanisms and pathogenicity. Journal of Nucleic Acids 2009; 138
Marsik C, Halama T, Cardona F, Wlassits W. Regulation of Fas (Apo-1, CD95) and Fas ligand expression in leukocytes during systemic inflammation in humans. Shock 2003; 20
Ma S, Chen GG, Lai PB. Bcl-2 family members in hepatocellular carcinoma (HCC): mechanisms and therapeutic potentials. Apoptosis cancer 2009; 7
El Bassiouny A, El-Bassiouni N, Nosseir M Zoheiry M. Circulating and hepatic Fas expression in HCV-induced chronic liver disease and hepatocellular carcinoma. Medscape J Med 2008; 10
Pawlotsky JM. Interpretation of virological tests for hepatitis C. Hepatology 2002; 36
Lok AS, Mahon BJ. Chronic hepatitis B: update of recommendations. Hepatology 2004; 39
Halfon P, Bourliere M, Penaranda G. Real-time PCR assays for hepatitis C virus (HCV) RNA quantitation are adequate for clinical management of patients with chronic HCV infection. J Clin Microbiol 2006; 44
Jodo S, Kobayashi S, Nakajima Y, Matsunaga T. Elevated serum levels of soluble Fas/APO-1 (CD95) in patients with hepatocellular carcinoma. Clin Exp Immunol 1998; 12
Morton RF, Hebel JR, McCarter RJ. Medical statistics. In: By: Morton RF, Hebel JR, McCarter RJ. A study guide to epidemiology and biostatistics. 5th ed. Gaithersburg, Maryland: Aspen Publication 2002; 71–74.
Mohamed A, Loutfy S, Craik JD. Chronic hepatitis c genotype-4 infection: role of insulin resistance in hepatocellular carcinoma. Virol J 2011; 8
Feier D, Platon ML, Stefanescu H, Badea R. Transient elastography for the detection of hepatocellular carcinoma in viral C liver cirrhosis. J Gastrointest Liver Dis 2013; 22
Zekri AN, El-din HM, Bahnassy AA. Serum levels of soluble Fas, soluble tumor necrosis factor-receptor II, interleukin-2 receptor and interleukin-8 as early predictors of hepatocellular carcinoma in Egyptian patients with hepatitis C virus. Comp Hepatol 2010; 95
Schmid M, Kreil A, Jessner W, Homoncik M. Suppression of haematopoiesis during therapy of chronic hepatitis C with different interferon alpha mono and combination therapy regimens. Gut 2005; 54
Giannini EG, Savarino V. Further insights into the causes of thrombocytopenia in chronic hepatitis C. J Gastrointest Liver Dis 2010; 4
Nawaz M, Siddique S, Manzoor I, Nadeem S. Infection of hepatitis C virus genotypes in hepatocellular carcinoma patients from rural areas of Faisalabad region, Pakistan. Afr J Biotechnol 2011; 10
Chor SY, Hui AY, Chan KK, Go YY. Anti-proliferative and pro-apoptotic effects of herbal medicine on hepatic stellate cell. J Ethnopharmacol 2005; 22
Talaat ZR, Desoky EA, Mona AH. Hepatitis C virus and apoptosis: the role of Fas (Apo-1/CD95) in chronic hepatitis C virus infection. Egypt J Immunol 2000; 7
Abdelgwad T, Badra GAbdelHafeez N. Expression Of Fas/Apo-1 (Cd95) In Patients With Hepatocellular Carcinoma. Journal of virology 2010; 2
Peng Z, Tang H, Ling Y, Han G. Apoptosis and Fas system are significantly involved in the process of liver cirrhosis converting into hepatocellular carcinoma. J TongiI Med Univ 2001; 221
Yoneyama K, Goto T, Miura K. The expression of Fas and Fas ligand, and the effects of interferon in chronic liver diseases with hepatitis C virus. Hepatol Res 2002; 24
Lee SH, Shin MS, Lee HS, Bae JH. Expression of Fas and Fas-related molecules in human hepatocellular carcinoma. Hum Pathol 2001; 32
Roskams T, Libbrecht L, Van B. Fas and Fas ligand: strong co-expression in human hepatocytes surrounding hepatocellular carcinoma: can cancer induce suicide in peritumoural cells? J Pathol 2000; 191
Hammam O, Mahmoud O, Zahran M, Aly S. The role of fas/fas ligand system in the pathogenesis of liver cirrhosis and hepatocellular carcinoma. Hepat Month 2012; 12
Morcos N, Khafagi E, Mogawer M, Ali M. Evaluation of biomarkers for the detection of hepatocellular carcinoma in patients with hepatitis C virus. J Exp Integr Med 2012; 2
Kern P, Dietrich M, Hemmer C. Increased levels of soluble Fas ligand in serum in Plasmodium falciparum malaria. Infect Immun 2000; 68
Valva P, De Matteo E, Galoppo MC. Apoptosis markers related to pathogenesis of pediatric chronic hepatitis C virus infection: M30 mirrors the severity of steatosis. J Med Virol 2013; 82
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]