Serum Alpha-Fetoprotein, Albumin and Previous Antiviral Treatment, Can Predict Non-Response to Direct Antiviral Therapy in Egyptian Patients with Chronic Hepatitis-C

Background & Aims: Direct acting antiviral therapies (DAAs), are currently the state of the art therapy of chronic Hepatitis C (CHC) giving hope particularly to patients with liver cirrhosis. The aim of the study was to investigate the ability to use baseline data as predictors of non-response to DAAs in patients with CHC. Methods: Baseline demographic and laboratory characteristics were collected for patients with CHC eligible for DAAs therapy. Patients were collected from March 2016 to October 2016 from Damanhur Viral Hepatitis Center (Boheira Governorate, Egypt). Monthly follow up was done during treatment to confirm safety, then at week 12 after the end of treatment to confirm sustained virological response (SVR) using routine laboratory data, ultrasonography, and quantitative HCV-PCR. Results: This observational study included 2446 patients with CHC who received DAAs (combined sofosbuvir and daclatasvir with or without ribavirin). Their mean age was 50 ± 9.5 year and 57.3% were females. About 47.4% were cirrhotic and 299 (12.2%) patients were treatment experienced. 96.24% patients achieved SVR-12. Baseline AFP was significantly higher in non-responders (14.3 ng/ml versus 9.5 ng/ml respectively, P-value < 0.001). Multivariate logistic regression analysis revealed that SVR-12 was significantly associated with being treatment naïve, having higher Albumin levels and having AFP level ≤ 10. Conclusions: The independent factors affecting SVR-12 were AFP level ≤ 10 ng, being treatment naïve, and serum albumin levels.


Introduction
Chronic hepatitis C (CHC) is considered a major etiology of chronic hepatitis and cirrhosis around the world particularly in Egypt [1,2]. Egypt previously had the highest Hepatitis C virus (HCV) burden worldwide as reported in 2008, with 90% of patients infected with genotype-4 [3,4]. During 2015, the sero-prevalence of HCV infection declined to 6.3% [5] with an overall estimated 30% reduction [2,6].
Patients received a single daily dose of sofosbuvir (400 mg/day) and daclatasvir (60 mg/day). RBV recommended dose was 1200 mg daily if the patient's weight was above 75 kg, and 1000 mg daily if the patient weight was less than 75 kg, given in two divided doses. It was added if patient is treatment experienced or one of criteria for difficult to treat is present; total bilirubin > 1.2, serum albumin < 3.5, INR > 1.2 or Platelets < 150000. Liver cirrhosis was diagnosed on clinical basis according to laboratory tests and findings of abdominal ultrasound and transient Elastography above 14 Kpa.

AFP assay
Quantitative assessment of serum AFP was done using the CanAg AFPEIA enzyme immunometric assay kit (Fujirebio Diagnostics AB, Göteborg, Sweden).

Follow up
Follow up was done every month during treatment to confirm safety, then at week 12 after the end of treatment to confirm sustained virological response (SVR). Follow up was performed using routine laboratory data, ultrasonography, and quantitative HCV-PCR to confirm SVR.

Statistical analysis
Data were summarized using descriptive statistics (mean and standard deviation). Number and percentage were used for qualitative data. Statistical difference between groups were done using chi square test for qualitative data, independent t-test for quantitative normally distributed data and Mann-Whitney for quantitative non-normally distributed data. Value 0.05 was selected as a significant level for the test. Those factors demonstrating significant association in bivariate analysis and any others believed to be important regardless of these results were included in a multivariate logistic regression model. Analyses were performed using SPSS V.

Results
The demographic features of the patients are shown in Table 1 with no significant difference among responders and non-responders.The age of the included patients ranged between 20-74 years with a mean of 50 ± 9.5 years. Females represented 57.3% of patients. A total of 299 (12.2%) patients had received previous antiviral therapy (treatment experienced), and a total of 1160 (47.4%) patients were cirrhotic.
Comparison of baseline lab results revealed no significant difference between responders and non-responders except for Albumin, AFP, WBC count, Platelet count and Prothrom-hepatitis. Elevated serum AFP is a marker for hepatocellular carcinoma (HCC) in patients with chronic liver disease [8,9]. CHC may lead to fluctuations in AFP that makes it difficult to differentiate from the development of HCC [10]. AFP was found to normalize after antiviral treatment with interferon (IFN) [11].
Over the past several years, DAAs have replaced IFN for the treatment of CHC that improved safety, tolerability and utility [12][13][14][15]. The goal of HCV treatment is the achievement of SVR with undetectable HCV-RNA by highly sensitive quantitative assays 12 weeks after treatment (SVR12) which is highly concordant with the previous SVR24 in the interferon era [16,17].
Non-response is often related to relapse, a rebound in HCV-RNA once therapy is terminated after being undetectable at end of therapy, and less frequent to viral breakthrough while on treatment. Non-response to treatment has been related to combinations of host, viral, and treatment-related factors [18]. This study aims to investigate the relation between baseline AFP and the response to treatment in 2446 chronic HCV patients treated with DAAs (combined sofosbuvir and daclatasvir with or without RBV) using routine pretreatment workup.

Patients and Methods
This study included 2446 Egyptian patients with treatment naive and treatment-experienced CHC who completed their combined antiviral treatment regimens. All patients were candidates for anti-viral therapy according to the guidelines of The National Committee for Control of Viral Hepatitis (NCCVH). The patients were recruited from Damanhur Viral Hepatitis Center (Boheira Governorate) affiliated to the NC-CVH within the period from March 2016 and October 2016. HCV infection in all patients was confirmed by positive quantitative PCR for HCV infection.
The study was performed in compliance with the ethics principles of the 1975 Declaration of Helsinki and its later amendments with good clinical practice (GCP) guidelines. All patients signed a written informed consent. The study was approved by the ethical committee of The National Committee for Control of Viral Hepatitis (NCCVH).

Exclusion criteria
Hepatic decompensation e.g. encephalopathy and/or ascites, calculated creatinine clearance ≤ 30 mL/min, extrahepat-which revealed that responders who achieved SVR-12 were significantly associated with being treatment naïve, having higher Albumin levels and having AFP level ≤ 10 (p < 0.05).

Discussion
This study included 2446 patients with chronic hepatitis C infection who received direct acting antiviral therapy. SVR-12 rate among these patients was 96.24%. We aimed to study the role of baseline serum AFP and other baseline patients factors as a predictor of response to treatment with DAAs.
The normal adult serum AFP concentration does not exceed 6 ng/ml. Elevations of serum AFP > 20 ng/ml were present in patients with HCV-related cirrhosis but without HCC with a prevalence ranging from 10% to 43%. AFP levels are influenced by non-tumoral factors such as etiology of chronic liver disease and progression of cirrhosis [20][21][22]. AFP is also bin concentration (p < 0.05). Treatment responders had sig-nificantly higher Albumin level, WBC count, Platelet count and Prothrombin concentration and significantly lower AFP level (p < 0.05) ( Table 2). The mean baseline serum AFP value was 9.7 ng/ml for all patients. Patients who did not achieve SVR showed significantly higher baseline AFP levels (14.3 ng/ ml versus 9.5 ng/ml, P value-< 0.001). SVR12 rate among these patients was 96.24%.
Higher levels of AFP were significantly associated with lower odds of being a responder at week 24. Patients with AFP ≤ 10 had 5.7 times higher odds to be responder com-pared to those who had AFP > 100. Patients with AFP ≤ 10 had two times higher odds to be responder compared to those who had AFP > 10 (Table 3). Table 4 shows the multivariate logistic regression analysis   In conclusion; high AFP more than 10ng, being a routine pretreatment laboratory assessment, is a predictor of non-response to DAAs in patients with chronic HCV genotype 4.

Conflict of Interest
All included authors declare absence of any financial or personal relationships with other people or organizations that could inappropriately influence and bias the work.

Submission Declaration
This work has not been published previously, is not under consideration for publication elsewhere, its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out and, if accepted, will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder.

Contributors and Authorship
All authors have contributed significantly to finish this work; all authors are in agreement with the content of the manuscript Design of the study: Mohamed Said Abdelaziz, YasminSaad Performance of management: Mohamed Said Abdelaziz, HosamDabes, KadriElSaeed, and YehiaElShazly raised in non-hepatic malignancies such as pancreatic, gastric, biliary and germ cell tumors [19].
For chronic hepatitis C patients, the HCV-coding core protein is known to upregulate the transcription of several molecules that activate the cell cycle and induce proliferation in hepatocytes, and it may also upregulate AFP transcription [23]. Therefore, mild elevation of the serum AFP level is sometimes seen in patients with chronic active hepatitis C but without HCC [19,24]. The percentage of chronic hepatitis C patients with an elevated AFP level (≥ 10 ng/mL) ranges from 11.6% to 43% [22,25,26].
Previous studies on CHC Egyptian patients, where genotype 4 is the prevalent genotype, revealed that serum AFP levels were found to be elevated in 12.6% of patients with 10 ng/ml upper limit of normal (ULN). Other studies reported elevated serum AFP with levels ranging between 10 and 30 ng/mlULN, with variable prevalence [27,28]. AFP used to be a strong predictor of response to the old standard of care therapy of combined pegylated interferon and ribavirin [29].
Studies have concluded that the SVR rate was higher among patients with serum AFP levels below rather than above the median value 5.7 ng/ ml [19,23] and have confirmed the value of serum AFP levels in predicting treatment outcome in CHC patients, regardless of the infecting genotype [19].
Serial AFP assay during and after treatment was not performed due to financial limitations. In addition, this serial AFP assay is not routinely performed in real life practice during