Evaluation of the Diagnostic Value of Serum Cytokeratin-8 as a Marker of Liver Injury in Chronic Hepatitis C Patients

Background: Hepatitis C virus (HCV) is considered the most health problematic in Egypt. Its severity ranges from mild illness to serious complications as cirrhosis and hepatocellular carcinoma. Keratins emerge as markers of liver injury beside significant contributors to liver disease pathogenesis. Aims: We analyzed the cytokeratin-8 serum levels and blood mRNA expression in chronic hepatitis C patients to evaluate serum CK8 role as a marker of liver injury. Patients and Methods: This study included 100 Egyptian patients with liver disease. They were 82 patients with chronic hepatitis C and 12 patients with hepatitis C-induced cirrhotic changes. Fifteen healthy controls were also included in the study. All studied subjects underwent a clinical assessment and complete laboratory evaluation. For patients groups a conventional abdominal ultrasonography and guided liver biopsy were performed with histopathological examination to assess the grade of inflammation and stage of fibrosis according to the Metavir scoring. The levels of CK-8 serum and blood mRNA expression were measured. Results: Serum CK-8 levels and mRNA expression were increased in HCV and cirrhotic patients compared to control group ( P <0.001*). Serum CK-8 levels were positively correlated with Metavir score in patients groups (r=0.714, P <0.001) (r=0.447, P <0.001) and in selected patients with inconclusive FIB4 index (values in between 1.45-3.25) (r=0.291, P =0.048) (r= 0.486, P <0.001). Conclusions: Serum CK 8 levels were positively correlated with Metavir score and FIB4 index. They may be useful for monitoring disease activity in chronic HCV patients especially with inconclusive FIB4 index.


INTRODUCTION
Chronic hepatitis C virus (HCV) infection is a significant public health problem in Egypt since there is no vaccine available, the response to usual treatment is limited. Globally, Egypt is believed to reach the highest HCV prevalence in the world [1]. HCV estimated prevalence in Egypt is 14.7% among general population in the year 2008 [2]. Liver responses to injury by fibrosis and ultimately it can give rise to liver cirrhosis which may lead to critical complications such as liver failure, hepatocellular carcinoma or lifethreatening esophageal and gastric varices. Liver cirrhosis and cancer represent the global causes of death [3][4].
Staging HCV infection is primarily depending on degree of histologic fibrosis in a liver biopsy sample.
However, the procedure is uncomfortable, painful, and costly. It carries a small risk for complication [5]. There is a trend to gain a benefit from utilizing an index based on serum fibrosis markers as the FIB-4 index [6][7][8] Mammalian cytoskeleton is mainly composed of microtubules, actin and intermediate filaments [9,10]. Keratins are the largest subfamily of cytoplasmic intermediate filaments contains more than 50 functional genes, which are expressed mostly in epithelial cells and skin appendages [11,12].
Keratins can be classified according to their tissue expression model into simple-epithelial keratins and keratins expressed in stratified epithelia and skin appendages [11]. Keratins include type-I keratin polypeptides (K9-K20) and type-II keratins 1-8 (K1-K8). Epithelial cells express at least one type-I and one type-II unique keratins which are both required to form obligate noncovalent heteropolymers [13,14].
Thus, hepatocytes are mainly sensitive to variation of keratin architecture and keratins appear as suitable markers of liver injury in addition to significant contributors to liver disease pathogenesis [12,15].
In this study, we investigated cytokeratin-8 serum levels and blood mRNA expression in patients with chronic hepatitis C to evaluate serum CK8 role as a marker of liver injury.

Study Design
This study was conducted on 100 patients with chronic HCV-related liver disease referred to the Internal Medicine Department. In addition, 15 normal volunteers with no clinical evidence suggestive of chronic liver disease were included as controls. The control population had normal liver biochemical profiles and was negative for anti-HCV antibodies. The duration of the study was 1 year.
The study was approved by local Investigational Review Board. The study design followed the Institutional Committee for the Protection of Human Subjects adopted by the 18th World Medical Assembly, Helsinki, Finland. Written informed consents were obtained from all participants in the study and confirmed by the IRB.
The patient inclusion criteria for this study included the following: Adult (23-58 years old), either gender with chronic HCV-related liver disease. Diagnosis was based on the presence of anti-HCV antibodies and HCV RNA in serum for at least 6 months in addition to the histopathologic evidence of chronic hepatitis or cirrhosis. Patients were excluded from the study if they were positive for HBsAg, had autoimmune or metabolic liver diseases, hyperlipidaemia, decompensated liver disease, currently or previously receiving IFNtherapy or their platelets count less than 100x10 3 /cm 3 .
All studied subjects received a clinical assessment and complete laboratory evaluation including fasting blood sugar level, complete blood count, liver biochemical profile, prothrombin time and activity , lipid profile, renal function tests, anti-nuclear antibodies (ANA), alpha fetoprotein (AFP). The hepatitis seromarkers HBsAg for HBV and anti-HCV were tested using ELISA (Abbott laboratories). The amount of HCV RNA in serum samples was quantified using a realtime PCR Step One instrument and software (Applied Biosystems).
For patients groups a conventional abdominal ultrasonography and guided liver biopsy were performed with histopathological examination to assess the grade of inflammation and stage of fibrosis according to the Metavir scoring system [16] and to exclude any combined liver insults. Serum samples at time of biopsy were used for serological analysis.
Liver biopsy requires using 16 gauge semiautomated biopsy needles. Liver specimens of 15 mm in length with minimal 4 portal tracts were fixed in 10% neutral formalin, then processed and embedded in paraffin. Sections were stained with haematoxylin and eosin, and Masson trichrome stains for detection of fibrosis.
Metavir scoring system demonstrated different stages of fibrosis (F0-F4) and grades of necroinflammatory changes (A0-A3) [16]. The histopathological examination of all the liver biopsies was performed by a single hepatology expert pathologist.

Calculated score
The FIB-4 index was calculated using Sterling's formula (7) as: Age (y) × AST (IU/L) /platelet count (×10 9 /L) ×√ALT (IU/L)) With regard to disease progression bias, the time interval between the determination of the FIB-4 index and liver biopsy was no longer than 7 days.
The patients were classified into the following groups:

Measurement of Serum Cytokeratin-8 & Genetic analysis of Cytokeratin-8 mRNA Expression
Serum levels of CK-8 were assessed using a quantitative measurement in serum using Sandwich ELISA(antibodies-online Inc. USA, Catalog no: ABIN817227).

Genetic analysis of cytokeratin-8 mRNA expression
Two ml blood in EDTA tubes obtained from peripheral blood of all subjects and RNA was extracted from samples using QIAamp RNA Blood Mini (QIAGEN GmbH, Hilden, Germany). All the RNA samples had an optical density 260/280 ratio ranging from 1.81 to 1.875, which confirms the good quality of the RNA. RNA samples with an optical density 260/280 ratio of less than 1.8 were excluded from the study.
The cDNA is used as a template to amplify the studied gene and it normalized using glyceraldehydes-3-phosphate dehydrogenase as housekeeping gene. The assay identification numbers of target and housekeeping genes are as followed: cytokeratin 8 (Assay ID #  Hs01595539_g1) and Glyceraldehyde 3phosphate dehydrogenase (GAPDH) gene (Assay ID # Hs99999905_m1) using the primers and Probes assays listed in TaqMan Gene expression assays from Applied Biosystems.
DNA amplification of the cytokeratin 8 gene was carried out by real time PCR using the real time PCR Step One instrument and software (Applied Biosystems). This included an initial denaturation step for 10 min at 95°C, followed by 40 cycles of annealing and elongation for 15 seconds at 95°C and 1 min at 60°C, respectively. Instrument raw data (fluorescence) for all the samples were converted into threshold cycles (Ct) using SDS 1.2 software (Applied Biosystems). Ct values were then imported onto an Excel worksheet for relative quantification (RQ).
For RQ calculation, the geometrical mean of GAPDH was used as a normalization factor.
Calculation of the results was performed by application of the comparative Ct method for RQ, 2 -∆∆Ct ∆Ct; unknown =Ct target -Ct reference The difference in threshold cycles for target and reference genes is as follows: ∆∆Ct =Ct unknown -Ct calibrator Where, ∆Ct, calibrator is the mean value for a healthy control human.

RQ=2 -∆∆Ct
The fold change, defined as the ratio between the averaged normalized expression levels of targets in neoplastic and corresponding nonneoplastic samples, was calculated. Normalized RQ values were log 2 transformed for statistical analysis.

Statistical Analysis
Statistical presentation and analysis of data in the present study was carried out; continuous data were expressed as mean±SD, whereas categorical data were expressed as number and percentage. Categorical data were compared using the X 2 test. Continuous data between two groups were compared using Student's t-test. Different parameters were compared using Pearson's correlation test. Statistical significance was defined as a P-value of less than 0.05. Analyses were carried out using the SPSS program, version 17 (SPSS Inc., Chicago, Illinois, USA) and the GraphPad Prism software (Graph-Pad Prism Software Inc., San Diego, California, USA).

RESULTS
The present study involved 82 patients with chronic hepatitis C and 18 patients with HCVrelated liver cirrhosis.
The laboratory data of studied groups and histopathological features of the patients groups were illustrated in Table 1, Figs. 1-2.
There was an increase in serum CK8 levels in HCV and cirrhotic patients compared to control group (P <0.001*). There was also an increase in serum CK8 levels in cirrhotic patients compared to HCV patients (P <0.001*). There was no difference in viremia degree between the two patients groups ((P >0.05) ( Table 1).
The relative expression levels of keratin 8 mRNA were found to be significantly upregulated, up to 7-fold in HCV patients, 10 fold in liver cirrhotic patients compared with normal controls. The cytokeratin 8 normalized mRNA levels in HCV and liver cirrhotic patients compared with normal controls were 7.5 (0.9-8.7), 10.5 (2.9-11.7) respectively, whereas in healthy controls it was 1.21 (0.5-4.1). Increased CK 8 mRNA expression was observed in 57 out of 82 HCV and 16 out of 18 cirrhotic patients, however there was no significant expression difference in comparison between HCV and cirrhotic groups Table 1.
There was a significant increased CK 8 mRNA expression with increased serum CK8 levels in both HCV and cirrhotic patients (P <0.001) Table 2.
There were positive significant correlations of serum CK8 levels with ALT, AST and FIB4 index, while there were negative significant correlations with platelet count and prothrombin activity (P < 0.05) Table 3.
There were positive significant correlations of serum CK8 levels with grade of inflammation (r=0.714, P <0.001) and stage of fibrosis (r=0.447, P <0.001) in patients groups (Fig. 3).
In ROC curve analysis serum CK8 gave significant data either when its levels compared between the patients groups or in different stages of fibrosis as following: when serum Ck > 6.9 (ng/mL), sensitivity, specificity positive predictive value, negative predictive value and AUC were 66.7, 96.3, 97.0,93.2, 87.2 respectively Table 4, Fig. 5.     Fig. 6). (2). The liver's response to injury by fibrosis. In the end this process can give rise to liver cirrhosis, which may result in the life threatening complications including portal hypertension, liver failure and hepatocellular carcinoma [17].

Chronic hepatitis C virus infection is a major health problem worldwide especially in Egypt
However, liver biopsy remains the gold standard for assessing presence, type and stage of liver fibrosis and for description of necroinflammation; it is a risky invasive procedure. Therefore, it cannot be carried out in many cases [18]. Mallory-Denk bodies (MDBs), are hepatocyte cytoplasmic inclusions present in several chronic liver diseases, including chronic hepatitis C (CHC) [12,19]. Keratin polypeptides 8 and 18 (K8/K18) are the main components of MDBs and expected to play a key cytoprotective function in the liver [12]. Keratin genes KRT8, KRT18 and KRT19 have been connected with increased liability to end-stage liver disease [20,21] and increased fibrosis in CHC [22].   Normally, the total amount of cellular CK8 is present at a stable level. The function of CK8/18 in the liver is protection from mechanical and non-mechanical stress which can lead to cell death. Stress can affect cytokeratins expression levels, expression profiles and posttranslational modification [12].
Our study was performed to examine the cytokeratin 8 serum levels and blood expression in patients with chronic HCV infection and evaluate its role as a marker for early liver injury by correlating serum CK8 level with severity of hepatic inflammation, fibrosis and FIB4 index.
Holmberg et al. [24] observed in their large observational cohort that FIB-4is a significant index to clinicians because it is simplify to calculate and easily available from hospital or clinic laboratories during usual patient care. Therefore, it would be helpful to screen patients who need biopsy and therapy.
Vallet-Pichard et al. [7] identified for values outside 1.45-3.25, the FIB-4 index is a simple, accurate and inexpensive method for assessing liver fibrosis.
However, FIB4 index reflect alterations in hepatic functions rather than in extracellular matrix (ECM) metabolism. Many HCV studies have identified normal transaminase levels in about 25%-30% of chronic HCV patients; there may be a potential advantage in assessing marker of direct liver injury that does not include transaminases [25].
In the present study, patients with chronic HCV had higher serum CK-8 levels than healthy controls. The highest level was observed in patients with cirrhosis.
This study also revealed increased expression of CK8 in both liver disease groups than the control group. There was also an increased expression in cirrhotic patients than chronic HCV patients however it did not reach a significant value.
This was in agreement with Ku et al. [15] who observed that there was an increased in expression of CK8/18 in response to liver injury by 3-fold, which may be a factor of the essential cytoprotection provided by CK8 and CK18 in the liver.
Their cytoprotection presented by an important role in mechanical stability of hepatocytes, and a target through toxic stress by induction of apoptosis/necrosis. That was observed by Tarantino et al. [26] who found an increase in level of tissue polypeptide-specific antigen, a serological mirror of keratin 18 in non-alcoholic steatohepatitis patients (NASH) compared to either pure fatty liver patients (FL) or healthy volunteers. They also observed that this marker is a better than alanine aminotransferase activity, ultrasonography or the combination of both parameters in differentiating NASH from FL.
Toivola et al. [27] found that in chronic HCV infection, CK8 phosphorylation is a dependable marker for liver disease progression (hyperphosphorylation) or regression (relative hypophosphorylation or return to basal phosphorylation). Furthermore, Strnad et al. [23] found that a number of CK8 gene variants are increased in patients with chronic HCV infection.
Sun et al. [28] found in their study that CK8 mRNA expression increased in HCV infected cell culture compared to other culture and suggested that HCV up-regulates CK8 expression in HCV cell culture cells, and that CK8 expression is significantly associated with HCV.
This study observed a significant increased CK 8 mRNA expression with increased serum CK8 level in both HCV and cirrhotic patients.
Our study also found significant positive correlations between serum CK-8 levels and ALT, AST, FIB4, grade of inflammation and stage of fibrosis in both HCV and cirrhotic patients.
Furthermore, the present study also identified significant positive correlations between serum CK-8 levels and both grade of inflammation and stage of fibrosis in patients had FIB4 index values inside 1.45-3.25.
This study identified increased serum CK8 levels with increased degree of inflammatory activity. These finding encourage a central role for apoptosis in disease pathogenesis, and this finding was similar to the results reported by Bantel et al. [29] and Fuentes-Gonzalez et al. [30]. They explained infection with HCV by inflammatory liver damage and a long viral persistence related with an increased risk of rising hepatocellular carcinoma. Apoptosis initiation upon HCV infection may seriously result in liver damage, while inhibition of apoptosis may contribute to HCV persistence and development of hepatocellular carcinoma.
Strnad et al. [23] revealed that there was an association between CK8/18 variants with development of liver fibrosis in chronic hepatitis C patients.
Wang et al [31] and Lee et al. [32] in their study explained that CK8/18-deficient animals had a marked susceptibility to tumor necrosis factor (TNF)-induced cell death and Fas-induced apoptosis. As CK8 and CK18 are in resistance to TNF family receptors-and Fas-induced apoptosis.
Xun et al. [33] reported that CK8 was participate in cytoprotective role against HCV infection and modulate the cellular response to proapoptotic signals to resist apoptosis and fibrosis. CK8 could be used as a marker to predict liver disease severity, treatment response and fibrosis.

CONCLUSION
In conclusion, the serum levels of CK-8 were observed to be equivalent to its blood expression. As a result, serum CK-8 levels could be used as a marker for disease progression and activity in HCV patients instead of the invasive tool current ''gold standard'' biopsy especially in patients with inconclusive FIB4index (values inside 1.45-3.25).