SVR Rates of HCV-infected population under PEG-IFN-α/R treatment in Northwest China

Chronic HCV Patients taking PEG-IFN-α/R from different ethnic groups have different probabilities of reaching a sustained viral response (SVR). There are many influence factors, such as HCV genotype, IL-28B single-nucleotide polymorphisms (SNP), Fibrosis 4 index (FIB-4), and aspartate aminotransferase-to-platelet ratio index (APRI) score. But the baseline factors in relation to treatment outcome was still not much clear. We evaluated data from 231 chronic HCV patients with or without liver fibrosis and their antiviral efficacy after treatment with pegylated interferon plus ribavirin (PEG-IFN-α/R) for 24–48 weeks. IL-28B SNP and HCV genotypes were analyzed with genome sequencing using pyrosequencing. Sustained viral response (SVR) rates of patients with HCV 1b and 2a genotypes were 52.25% (58/111) and 75.28% (67/89) (P < 0.01). SVR rates of patients with IL-28B rs8099917 TT, rs12979860 CC and rs12980275 AA were 92.41% (25/27), 92.86% (26/28) and 88.89% (24/27) separately. We found that SVR rates in HCV 1b and 2a patients were only 31.0 and 39.4% if their FIB-4 > 3.25. In addition, when their APRI > 2, only 30.3% of HCV 1b patients and 50.2% of HCV 2a patients could obtain SVR. There were high proportion of HCV genotype 1b and 2a in Northwest China. In both HCV 1b and 2a genotypes, patients with protective-genotype of IL-28B were more likely to obtain SVR. However, those with significant fibrosis or cirrhosis were less likely, no matter their genotype. Combined factors of HCV genotype, IL-28B genotype, FIB-4 and ARPI may indicate high prediction and clinical value regarding treatment with PEG-IFN-α/R and prognostic evaluation of chronic hepatitis C patients.


Background
Hepatitis C is an infectious disease that is widely spread geographically. It has been reported that about 180 million people are infected with the hepatitis C virus (HCV) worldwide, accounting for about 3% of the current population [1]. The incidence of infection can be easily neglected and thus may develop into cirrhosis and even HCV-related hepatocarcinoma and liver failure, which pose serious threats to human health. More than 0.35 million people have died from HCV-related liver disease [2]. The application of pegylated interferon (PEG-IFN)-alpha combined with ribavirin (PEG-IFN-α/R) had been considered to be the most popular and effective therapy in blocking virus replication before 2015 [3]. While the novel sofosbuvir-ledipasvir opened a new time for treatment of eligible HCV-infected patients with 90-100% efficiency [4,5]. Unfortunately sofosbuvir is much more expensive, with an estimated cost of an additional $65 billion during the next 5 years [6]. It should be carefully thought about the necessity and how much people have to take DAA drugs. And it is not the time to ignore the application of PEG-IFN-α/R, especially in China.
Patients taking PEG-IFN-α/R from different ethnic groups have different probabilities of reaching a sustained viral response (SVR). In the United States, the rate of obtaining SVR in the black population is almost 50% less than of white patients, and the probability of obtaining SVR in white patients with HCV genotype 1 treated by PEG-IFN-α/R is approximately 42-53% [7]. In China, the SVR rate was a light higher, could reach 65.3% [8]. We recently found that northwest patients of China seem to have higher SVR that was 72.6%. Because there were seldom immigrant in the northwest population, the high SVR is reasonable caused by geographic specificity, such as genotype of HCV and polymorphisms of IL-28B.
Determination of HCV genotype is important to predict response to antiviral therapy and time of treatment [9]. Early clinical trials have reported that SVR rates for patients with genotype 1 (42-46%) are lower than rates for patients with non-type 1 genotype (76-82%) [10]. On the other hand, genome-wide association studies have suggested that host IL-28B single-nucleotide polymorphisms (SNP) rs12979860 CC and rs8099917 TT are significantly correlated with SVR in patients receiving PEG-IFN-α/R treatment [11]. The probability of obtaining SVR is further reduced in vivo if IL-28B is mutated in patients with HCV genotype 1 infection [12,13].
Liver fibrosis had been suggested being closely associated with the risk of HCC development in chronic hepatitis C patients [14]. The eradication of HCV with antiviral therapy will prevent the progression of chronic hepatitis and associated complications [15]. But it was never paid more attention in China [8].
To determine the favorable patients for treatment with PEG-IFN-α/R, HCV genotype, IL-28B SNP, Fibrosis 4 (FIB-4) index and aminotransferase-to-platelet ratio index (APRI) score we retrospectively analyzed with SVR in this study.

Patients
All patients with CHC were enrolled for this research with signed informed consents following the protocols approved by the Institutional Review Board of the Fourth Military Medical University (Table 1). Inclusion criteria: patients were firstly diagnosed as HCV infection since August 2013, naive-treatment from October 2013 and HCV RNA > 1000 IU/mL. Exclusion criteria: Patients with recurrence of hepatitis C, hepatitis infected with HAV, HBV, HDV, HEV, EBV or CMV; HIV infection, Diabetes, autoimmune liver disease and HCC etc. were excluded.
We collected data on 230 patients with chronic HCV infection who were seen at Xijing Hospital from October 2013 to February 2016. The average age was 46.71 years (range, 20-80 y), with 112 male and 118 female patients. Patients had been treated with standard of care for 24-48 weeks. PEG IFN-α/RBV: The recommended dose of PEG IFN-α-2a (Pegasys Roche Shanghai) for chronic hepatitis C was 180 ug per time, once a week, subcutaneous injection of the abdomen or thigh. The dose of RBV was determined by the genotype of virus: the dose for genotype 2 or 3 was 800 mg a day for 24 weeks, and the dose of genotype 1 was 1000-1200 mg daily according to body weight, for 48 weeks. We mainly investigated outcomes after 24 weeks.
There are many serological markers for HCV or evidence of liver disease including HCV RNA was used to understand the activity of virus replication, ALT, AST, Total bilirubin, direct bilirubin, indirect bilirubin, albumin, globulin, choline esterase, alkaline phosphatase, phosphatase and abdominal ultrasound were used for evaluation of liver damage, CT or MRI was used to be clear of the extent of liver damage. Liver biopsy is the gold standard for evaluation of liver inflammation and The low number of patients with 2b, 4a, 5a and 6a did not meet the statistical requirements; therefore, these were excluded for follow-up statistics fibrosis staging in patients. In addition, there were also patient compliance issues. After careful consideration we chose these common and easy to get markers as ALT and AST for liver damage, FIB-4 and APRI to evaluate liver fibrosis. Use of viral content to evaluate curative effect was according to the 2014 European Association for the Study of the Liver Recommendations on Treatment of Hepatitis C and the Guideline of Prevention and Treatment of Hepatitis C [16,17]. We evaluated SVR using quantitative real-time fluorescence polymerase chain reaction (ViiA7 OX, Life Technology) of HCV RNA (less than 15 IU/mL) for at least 24 weeks of follow-up at the end of treatment. Samples were collected and separated from the peripheral blood and serum and then stored at −20°C until analyses.

DNA/RNA extraction
For DNA extraction for IL-28B gene detection, we used a blood genomic DNA extraction kit (Tiangen, Beijing, China). For HCV RNA extraction, we used the MinElute column QIAamp method and the virus genome DNA/RNA extraction kit (Tiangen). All extracted DNA/RNA were then immediately used for gene detection or stored at −80°C.

Gene sequencing
IL-28B gene polymorphism was detected with the use of a pyrosequencing method on Q24MDX (Qiagen, Hilden, Germany). Sequencing primers were designed by the Q24 PyroMark. HCV polymorphism sequencing primers were also provided by Qiagen.

Liver fibrosis staging
The degree of liver fibrosis was evaluated with APRI score, which can be used for the assessment of liver cirrhosis [18]. APRI scores > 2 in adults indicate that the patient has already had liver cirrhosis. The APRI score is calculated as follows: (aspartate aminotransferase [AST]/ULN) × 100/ platelets (10 9 /L), where ULN is the upper limit of normal value. Fibrosis-4 index was based on values of ALT, AST, platelet count and patient age. This index can be used to diagnose liver fibrosis (similar to least significant fibrosis using METAVIR F scoring system ≥2) [19]. A significant liver fibrosis has occurred if a patient shows aFIB-4 index of >3.25. FIB-4 is calculated as follows: age × ALT (IU/L)/ (platelet count [10 9 /L] × AST [IU/L]) 1/2 .

Statistical analyses
We used Pearson chi-squared and Kruskal-Wallis tests to analyze the qualitative data with SPSS 19.0 (IBM, USA.). A logistic regression model was used to analyze the correlation between SNPs (IL-28B rs8099917, IL-28B rs12979860 and IL-28B rs12979860) and SVR of patients. Odds ratio (OR) was used to describe the correlating degree of disease and exposed factors. OR tests were two-sided tests, in which P < 0.05 was considered to be statistically significant.

HCV genotype and distribution
HCV genotypes of 230 patients with chronic HCV infection were sequenced, with results shown in Table 2. One patient had mixed infection of 1b and 5a. For statistical analyses, this patient was analyzed in both the 1b and 5a genotype groups. Statistical results of 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a and 6a genotypes are presented in Fig. 1

IL-28B genotype in patients with chronic HCV infection
Fifty-one patients with HCV were included in the IL-28B gene polymorphism loci analyses (Table 3). Of the IL-28B genotypes, 76.47% of patients were  Table 3. Statistical results showed no statistical differences in age, sex, viral load and ALT and AST levels (P > 0.05).

Role of IL-28B SNP and HCV genotype in antiviral efficacy
We combined the results of IL-28B SNP and HCV genotype and analyzed the correlation between these factors. We found that 12/51 patients with HCV genotype 1b infection obtained SVR, in which 8/12 cases were rs12979860 CC (shown in Fig. 2c). We also found that the 7 of10 patients infected with HCV genotype 2a who obtained SVR were rs12979860 CC.  Tables 4 and 5.

Discussion
China has shown a new trend in HCV infection, with epidemic levels of genotypes1, 2, 3 and 6 and no genotypes 4 or 5 found. The most common genotype in China is1b and 2a, with incidence rates of 73.1 and 18.5%, followed by genotypes 3a, 6a, 3b and 1a. Genotypes 3 and 6 are geographically distributed more widely [20]. In our group, which included 230 patients with chronic HCV infection, sequencing results showed that the incidence of HCV in the Shaanxi region was > 86% with genotypes 1 and 2, with much lower numbers with genotypes 3 and 6, and none with genotype 4. More patients had genotype 1b (48.05%) and 2a (38.53%), followed by 1a, 3a, 3b, 2b and 6a. The high percent of genotype 2a might be one important reason for high SVR. And we truly found that patients with genotype 2a had greater SVR rates (75.28%) than patients with genotype 1b (52.25%). The results have a slight discrepancy versus the previous reports, which SVR rates for patients with genotype 1 were 42-46% and non-type 1 genotype were 76-82% [10,[21][22][23]. Furthermore, our SNP analysis results of 51 patients with chronic HCV infection (IL-28B rs8099917, IL-28B rs12979860 and IL-28B rs12980275 SNP) and sequencing results of 230 patients with chronic HCV infection showed that more patients had IL-28B rs8099917 TT versus rs8099917 GG, more had IL-28B rs12979860 CC versus TT, and more had IL-28B rs12980275 AA versus GG in Northwest China, similar to some previous reports [24,25]. Presence of rs8099917 is one of the independent predictors in HCV 1b patients treated with PEG-IFN-α/R or interferon-α 2 only. Presence of rs12980275 has great relevance with SNP of rs12979860 [26], which plays an important role in the prediction of SVR in HCV patients treated with PEG-IFN-α/R, particularly in patients with HCV genotype 1 or 4 [10,27]. So rs8099917TT, rs12979860CC, and rs12980275AA were considered to be protective genotypes [28]. In this study, SVR rates of patients with IL-28B rs8099917 TT, rs12979860 CC and rs12980275 AA were 92.41% (25/27), 92.86% (26/28) and 88.89% (24/ 27) separately. SVR rates in patients with protective genotypes accounted for more than sixty percent of total, whereas the SVR rates of non-protective genotypes were very low. This suggested that the protective IL-28B genotypes were more likely to result in patients with chronic HCV infection obtaining SVR, which is consistent with previous reports [26][27][28][29]. In addition, we corrected for age, sex, AST and ALT levels, HCV genotype and other factors with the use of SNPStats software [30] and found that patients with chronic HCV infection who obtained SVR 24 weeks after standard of care antiviral treatment were closely related to HCV genotype and IL-28B SNP (P < 0.05). That means more than sixty percent of patients are suitable for PEG-IFN-α/R treatment. Well, the major limitation in this part of study is that the sample amount was relatively too small to determine the role of IL-28B gene polymorphism in antiviral efficacy. However, our findings were in accordance with previous data [26][27][28][29].
Interestingly, we found that differences in cirrhosis progression between 1b and 2a were statistically significant, confirming that HCV genotype can influence the progression of liver cirrhosis. FIB-4 index > 3.25 or APRI score > 2 indicated that patients had significant liver fibrosis or even cirrhosis [31]. In this study, the SVR rates of patients with FIB-4 index > 3.25 and genoty-pes1b and 2a were 31.0 and 39.4%, respectively, which were much lower than that shown in patients with genotype 1b (62.06%) and genotype 2a (74.54%) with FIB-4 index ≤ 3.25. This indicated that patients with obvious liver fibrosis were less likely to reach SVR, which was associated with HCV genotype (1b or 2a). In addition, SVR rates of patients with APRI score > 2 were 30.3% for genotype 1b and 50.2% for genotype 2a. The large difference indicated that patients with cirrhosis had greater difficulty reaching SVR, for both HCV genotype 1b and 2a, and the progression of cirrhosis (APRI > 2) could be influenced by genotype. The clearance of HCV in patients with advanced-stage liver fibrosis can reduce the incidence of decompensated liver cirrhosis.

Conclusions
There were high proportion of HCV genotype 1b and 2a in Northwest China. In both HCV 1b and 2a genotypes, patients with protective-genotype of IL-28B were more likely to obtain SVR. However, those with significant fibrosis or cirrhosis were less likely, no matter their genotype. Combined factors of HCV genotype, IL-28B genotype, FIB-4 and ARPI potentially have a very high prediction and clinical value regarding treatment with PEG-IFN-α/R and prognostic evaluation of chronic hepatitis C patients.