Role of Plasma Osteopontin Level as a Predictor of Hepatic Fibrosis Regression and Response to Antiviral Treatment in Patients with Chronic HBV or Chronic HCV Infection

Background: Hepatitis B virus and Hepatitis C virus infection is one of the public health problems in Egypt. So we aimed to evaluate the efficacy of serum osteopontin as predictor of hepatic fibrosis regression and virological response in patients with chronic HBV or HCV infection. Methods: This study has been conducted on 74 HBeAg + ve chronic HBV infection, 74 chronic HCV infection and 74 healthy controls. HBV patients treated with Entecavir. HCV patients treated with sofosbuvir, daclatasvir with or without ribavirin. One year post HBeAg seroconversion and 3 months after end of regular antiviral treatment for patients with chronic HBV and chronic HCV infection respectively, hepatic condition was reevaluated. Results: 14.9% of patients with HBV, failed to achieve undetectable HBV DNA or HBeAg seroconversion and 2.7% of patients with HCV infection, failed to achieve SVR. In chronic HBV, pretreatment high serum osteopontin predict failure of virological response and hepatic fibrosis regression at a cutoff > 115.5, with 90.91% sensitivity, 82.54% specificity. Also high degree of liver stiffness predicts failure of hepatic fibrosis regression at a cutoff > 8.7, with 81.8% sensitivity, 73% specificity. Conclusions: In chronic HBV infection low osteopontin predicts good virological response and hepatic fibrosis regression. But it has no role in predicting SVR or hepatic fibrosis regression in chronic HCV infected patients.


Introduction
Egypt has the highest prevalence of hepatitis C virus (HCV) infection worldwide [1].Chronic hepatitis C virus infection is the main cause of end-stage liver disease with liver cirrhosis and hepatocellular carcinoma in Egypt, it's responsible for chronic hepatitis in about 80% of the infected patients, of them about 20% develop liver cirrhosis of them about 6% develop decompensated liver disease and 5% of the patients with liver cirrhosis develop hepatocellular carcinoma [2].
Sofosbuvir, daclatasvir, with or without ribavirin combination are safe and effective in treatment of chronic hepatitis C virus infection in Egypt [3].
Sustained virological response after 3 months of regular treatment achieved in about 95% of patients in the easy-to-treat group who received Sofosbuvir and daclatasvir, and reach about 92% in the difficult-to-treat group who treated with Sofosbuvir, daclatasvir, and ribavirin [3].
Direct acting antiviral (DAA) combinations proved to cause obvious improvements in hepatic laboratory parameters with good clinical outcomes in chronic hepatitis C virus treated patients [4].
The side effects reported post treatment with sofosbuvir, daclatasvir, and ribavirin combination range from mild adverse events as anemia, fatigue, headache, and itching to severe side effects as hepatocellular carcinoma and hepatic encephalopathy which reported in patients with advanced liver disease [5].

Hepatitis B virus (HBV) infection is one of the public health problems in
Egypt [6].
About 350 million patients in Egypt have chronic hepatitis B virus infection and more than 1 million die from liver cirrhosis and hepatocellular carcinoma annually [7].
The aim of therapy of patients with chronic hepatitis B virus infection is to preventthe liver disease progression by suppressing hepatitis B virus DNA replication [8].
The available oral antiviral drugs for treatment of chronic HBV infected patients include nucleos(t)ide analogue as lamivudine, adefovir, entecavir, telbivudine and tenofovir [9].
Effective antiviral treatment of chronic hepatitis B virus infection reduced the risk of long-term complications and improved patient survival [9].
Entecavir is a cyclopentyl guanosine analogue with a potent inhibitory effect on HBV DNA replication [10].
Entecavir is effective in the treatment of patients with either HBeAg positive or HBeAg negative chronic hepatitis B [11].
Treatment with Entecavir cause viral suppression with significant improvement in hepatic fibrosis and liver functions biochemical parameters [12].
Osteopontin, is a secreted Phosphoprotein involved in different hepatic pathological conditions such as inflammation, immunity, angiogenesis, liver fibrosis progression, invasion and metastasis of HCC [13].Open Journal of Gastroenterology In the liver, Osteopontin exists normally in the epithelial bile duct [14].
Osteopontin pathologically expressed in Kupffer cells, hepatic macrophages and hepatic stellate cells in areas of liver necrosis [15].
Osteopontin has a role in many liver diseases as acute liver cell failure [16], nonalcoholic fatty liver diseases [17], alcoholic liver diseases [18], hepatic fibrosis with chronic hepatitis B [19], and chronic hepatitis C [20].Also, Osteopontin considered as a better prognostic marker for early hepatocellular carcinoma compared with alpha-fetoprotein [21].
Hepatic fibrosis is the most dangerous complication of chronic hepatic disease that finally leads to cirrhosis with subsequent complications [22].
So, in this study we try to evaluate the effect of antiviral treatment in decreasing serum Osteopontin level as a marker of improving hepatic fibrosis and as a pretreatment marker to predict response to treatment in patients with chronic hepatitis B or chronic hepatitis C virus infection.

Aim of the Work
The current study aimed to evaluate the effect of antiviral treatment in decreasing serum Osteopontin level as a marker of improving hepatic fibrosis and as a pretreatment marker to predict response to treatment in patients with chronic hepatitis B or chronic hepatitis C virus infection.

Patients and Methods
This prospective comparative case-control study has been conducted on 222 subjects of them 74 naive patients with HBeAg positive chronic HBV infection with different degree of hepatic fibrosis (F0, F1, F2, F3, ≥F4), and 74 patients with chronic hepatitis C virus infection with variable degree of liver stiffness and 74 age and sex matched healthy control for assessment of normal serum osteopontin.All patients attend out patients' clinic of Tropical Medicine and Gastroenterology Departments during the period from July 2017 till August 2018.
All patients with chronic HBV infection were treated with Entecavir 0.5 mg or 1mg once daily oral on empty stomach according to hepatic condition.Patients were treated according to EASL 2017 guidelines for HBV infection treatment [23].
Patients with chronic HCV infection were treated with DAAS in the form of sofosbuvir 400 mg and daclatasvir 60 mg with or without ribavirin (800 -1200 mg) once daily for 12 weeks.Patients were treated according to the Egyptian protocol for treatment of chronic hepatitis C virus infection, which follow EASL 2015 guidelines for treatment of HCV infection [24].
At base line all included patients with chronic HBV infection or chronic HCV infection subjected to complete history taking, full clinical examination, liver function tests, liver fibrosis assessment (F0 -≥F4) using fibroscan, and serum Osteopontin measurement as a marker of liver fibrosis.
One year post HBeAg seroconversion and 3 months after end of regular anti-viral treatment for patients with chronic HBV infection and chronic HCV infection respectively, all patients are reevaluated clinically, laboratory and radiological for reassessment of improvement in hepatic condition.
Patients with HBV and HCV co-infection, HBeAg negative chronic hepatitis B, hepatitis D virus infection, human immunodeficiency virus, malignancy anywhere in the body, triphasic CT imaging of hepatocellular carcinoma (HCC) or history of HCC, excessive alcohol consumption more than 21 drinks per week for men and more than 14 drinks per week for women, autoimmune diseases, any liver diseases other than chronic hepatitis B or chronic hepatitis C virus infection, diabetes mellitus, kidney, heart or chest diseases, pregnancy were excluded from the study also chronic hepatitis C virus infected patients with high serum bilirubin (>mg/dl), low serum albumin level (<2.8 gm/dl), low platelet count (<50 × 10 3 /µl) with marked impaired INR (≥1.7) were excluded from the study as they were out of the treatment protocol.

The Laboratory Tests
1) The platelet count was done by Celtak haematology analyzer (Nihon Kohden, Japan).
2) The liver function tests including (total and direct bilirubin, serum albumin, AST and ALT) were done by BT 1500 full automated chemistry analyzer (Bioticnica, Italy).
3) The prothrombin time, prothrombin concentration and INR was done using Thromborel S kits (Siemens, Germany) using BE coagulator (Germany).Probability (p-value) p-value < 0.05 was considered significant.

Statistical Analysis
p-value < 0.001 was considered as highly significant.

Results
The present study included 222 subject classified to 3 groups, the first group include 74 apparent healthy control, the second group include 74 naive chronic HBV infected patient with their mean ages were 50.4 ± 9.1; 35 (47.3%) females and 39 (52.7%) males of them 25 (33.8%) had mild viral load, 25 (33.8%) had moderate viral load and 24 (32.4%) had high viral load.
The pretreatment laboratory data, mean serum osteopontin level and mean hepatic fibrosis level of our included patients with chronic hepatitis B and chronic hepatitis C virus infection were discussed in Table 1.
In patients with chronic HBV infection, Enticavir 0.5 mg/day or 1 mg/day reviles that, 11 (14.9%) of patients failed to achieve undetectable HBV DNA by real time PCR or HBeAg seroconversion.However, 2 (2.7%) of patients with chronic HCV infection failed to achieve SVR three months after end of regular Open Journal of Gastroenterology antiviral therapy with sofosbuvir 400 mg and daclatasvir 60 mg with or without weigh-based ribavirin (800 -1200 mg) once daily.
By comparing laboratory data and liver stiffness before and after therapy in patients with chronic hepatitis C virus infection, there was statistically significant difference (p-value < 0.05) between (Bilirubin and Platelets) and there was highly statistical significant difference (p-value < 0.001) between (AST, ALT, Liver stiffness and osteopontin) before and after therapy.
In patients with chronic hepatitis B virus infection, there were statistically significant difference (p-value < 0.05) between (liver stiffness and Platelets) before and after therapy while there was highly statistical significant difference (p-value < 0.001) between (AST, ALT, Bilirubin, viral load and osteopontin) before and after therapy (Table 2).
In patients with chronic HCV no statistical significant difference (p-value > 0.05) between responders and non-responders to therapy as regard laboratory data and liver stiffness before therapy.
In patients with chronic hepatitis B virus infection, by comparing between responders and non-responders to therapy as regard pretreatment laboratory data (AST, Bilirubin, Albumin, Platelets, viral load and liver stiffness) showed statistically significant difference (p-value <0.05), while there was post treatment highly statistical significant difference (p-value < 0.001) between responders and non-responders to therapy as regard pretreatment laboratory data (INR and osteopontin).This is illustrated in (Table 3).
The mean pretreatment osteopontin level in patients with chronic HBV, HCV and control were 82.3 ± 45.4, 78.7 ± 48.02 and 13.2 ± 6.4 respectively with osteopontin level range from 7 -31 ng/ml in apparent healthy control.
Using roc curve, it was shown that pretreatment high serum osteopontin can be used to predict failure of post treatment virological response to antiviral therapy and failure of achieving hepatic fibrosis regression in patients with chronic hepatitis B virus infection at a cutoff level of >115.5 ng/ml, with 90.91% sensitivity, 82.54% specificity, 83.9% PPV and 90.1% NPV.
Also using roc curve, it was shown that pretreatment high degree of liver stiffness value as evaluated by hepatic fibroscan can be used as a marker to predict failure of post treatment response to antiviral therapy in addition to failure of hepatic fibrosis regression in patients with chronic hepatitis B virus infection at a cutoff level of >8.7, with 81.8% sensitivity, 73% specificity, 75.2% PPV and 80% NPV (Table 4, Figure 1 & Figure 2).
However, in patients with chronic hepatitis C no role of pretreatment serum osteopontin to predict post treatment SVR or improvement in hepatic fibrosis.

Discussion
In humans Osteopontin is located on chromosome number 4, region 22 and it's composed of about 300 amino acids [25].
Osteopontin secretion is a marker to predict hepatic fibrosis in patients with hepatitis B virus or hepatitis C virus infection [26].In our study we try to evaluate the role of plasma Osteopontin level as a predictor of hepatic fibrosis regression and as a pretreatment marker that predict response to antiviral therapy.
In this study all included patients with chronic hepatitis B virus infection and chronic hepatitis C virus infection show marked improvement in liver function test, increase in platelets count and significant decrease in liver stiffness one year post HBeAg seroconversion and 3 months after regular antiviral therapy.
This come in agreement with Gai & Wu, 2017 who found that there were marked improvement in liver function and reduction in liver fibrosis post Entecavir antiviral therapy in patients with chronic hepatitis B virus infection [27].osteopontin may directly promote HCV replication [35].
In our study we have significant higher level of pretreatment serum osteopontin in non-responder patients compared with responder one.Also we found that low pretreatment serum osteopontin has a high efficacy in predicting post treatment hepatic fibrosis regression and effective virologicl response to entecavir antiviral therapy with undetectable HBV-DNA and HBeAg seroconversion in patients with chronic hepatitis B virus infection at a cutoff level of <115.5 ng/ml, with high sensitivity and specificity.
This come in agreement with Liu et al., 2017 who reported that low serum osteopontin may be a predictor of good prognosis in patients with chronic hepatitis B virus infection but high serum osteopontin directly correlate with liver cell injury [37].
In this study we also found that pretreatment low degree of hepatic fibrosis is a good marker that predicts post treatment hepatic fibrosis regression in chronic hepatitis B virus infected patients at a cutoff level of <8.7, but with lower sensitivity and specificity compared to serum osteopontin.
This come in agreement with Chon et al. 2017 who concluded that low pre-Open Journal of Gastroenterology treatment liver stiffness value (<12.0 kPa) was a significant predictor of fibrosis improvement in patients with chronic hepatitis B virus infection [38].
In conclusion: pretreatment low serum osteopontin in addition to early hepatic fibrosis were predictor of good prognosis in patients with chronic HBV infection with good virological response and hepatic fibrosis regression.Although, pretreatment serum osteopontin level was higher in chronic hepatitis C virus infected patients than post treatment one.But it had no role in predicting post treatment SVR or hepatic fibrosis regression.Further studies on large number of patients are needed to evaluate its role as prognostic marker in chronic hepatitis C virus infected patients.

3. 1 . 1
Sampling 8 ml of venous blood were collected from each patients under aseptic precautions and divided in 4 tubes: 1) 2 tubes containing EDTA (sterile EDTA containing Vacutainer) one for platelet count used directly and the other for HBV-DNA centrifuged, separated in sterile tube and frozen at −20˚C till the time of the assay.2) Plain glass tube for liver function tests, then after clotting, the tube was centrifuged at 3000 rpm for 5 min and then the serum was separated to be used for estimation of liver function (including liver enzymes, albumin, bilirubin-total and direct) and serum Osteopontin.3) Sodium citrate containing tube (Vacutainer) for prothrombin time, prothrombin concentration and INR.

4 )
HBV-DNA was done by StepOnePlus TM Real-Time PCR System (Thermo-Fisher Scientific, USA) using Taqman PCR mastermix (ThermoFisher Scientific, USA) after DNA extraction by Qiagen kit (Qiagen, Inc., USA) and according the manufacture's pamphlet.5 ml of venous blood obtained from all subjects in plain tube and centrifuged then the serum is separated for the laboratory tests: 5) HCV antibodies and HBsAg is done by the MiniVidas fully automated immunoassay system (Biomerieux, France).6) Osteopontin is estimated by Abcam's osteopontin human ELISA (En-zyme-Linked Immunosorbent Assay) kit is an in vitro assay for the quantitative measurement of human osteopontin in serum and plasma.This assay use an antibody specific for human osteopontin coated on a 96-well plate.Standards and samples are pipetted into the wells and osteopontin present in a sample is bound to the wells by the immobilized antibody.The wells are washed and biotinylated anti-human osteopontin antibody is added.After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells.The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of osteopontin bound.The stop solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Figure 1 .
Figure 1.ROC curve between responder and non-responder as regard serum osteopontin in patients with chronic hepatitis B virus infection.

Figure 2 .
Figure 2. ROC curve between responder and non-responder in patients with chronic hepatitis B infection as regard liver stiffness.

Table 1 .
Demographic data and baseline laboratory data of patients with chronic hepatitis B and chronic hepatitis C.

Table 2 .
Comparisons between laboratory data before and after therapy in patients with chronic hepatitis C infection and chronic hepatitis B infection.

Table 3 .
Comparisons between responders and non-responders as regard laboratory data before therapy in patients with chronic hepatitis C infection and chronic hepatitis B infection.

Table 4 .
Diagnostic performances of serum osteopontin and liver stiffness in discrimination of responder and non-responder of patients with chronic hepatitis B virus infection.