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IL28B polymorphism as a predictor of antiviral response in chronic hepatitis C

AIM: To evaluate the effect of single nucleotide polymorphisms of interleukin (IL)-28B, rs12979860 on progression and treatment response in chronic hepatitis C. METHODS: Patients ( n = respectively ( P < 0.001). Significant differences were found in the insulin resistance index ( P = 0.01) between patients with and without steatosis. Patients with the CT allele and absence of a SVR had a higher incidence of requiring threshold dose reduction of interferon ( P = 0.07). CONCLUSION: IL28B variation is the strongest host factor not related to insulin resistance that determines outcome of antiviral therapy. Baseline platelet count predicts the outcome of antiviral therapy in CT allele patients. Clinical aspects of the impact of IL28B SNPs on the efficacy of antiviral therapy and progression of CHC are not fully recognized. In the present study, potential factors influencing the efficacy of therapy in relation to IL28B polymorphism were investigated. reduction of IFN and decreased chance of reaching the SVR. This study demonstrated a new char-acteristic of IL-28b in CHC.

vs TT allele: odds ratio (OR), 25; CC vs CT allele: OR, 5.4), inflammation activity (G < 1 vs G > 1: OR, 3.9), fibrosis (F < 1 vs F > 1: OR, 5.9), platelet count (> 200 × 10 9 /L vs < 200 × 10 9 /L: OR, 4.7; OR in patients with genotype CT: 12.8), fatty liver (absence vs presence of steatosis: OR, 4.8), insulin resistance index (< 2.5 vs > 2.5: OR, 3.9), and baseline HCV viral load (< 10 6 IU/mL vs > 10 6 IU/mL: OR, 3.0). There was no association with age, sex, aminotransferases activity, body mass index, waist/hip ratio, or percentage body fat. There was borderline significance (P = 0.064) of increased fibrosis in patients with the TT allele, and no differences in the insulin resistance index between groups of patients with CC, CT and TT alleles (P = 0.12). Spearman's rank correlation coefficient between insulin resistance and stage of fibrosis and body mass index was r = 0.618 and r = 0.605, respectively (P < 0.001). Significant differences were found in the insulin resistance index (P = 0.01) between patients with and without steatosis. Patients with the CT allele and absence of a SVR had a higher incidence of requiring threshold dose reduction of interferon (P = 0.07).
CONCLUSION: IL28B variation is the strongest host factor not related to insulin resistance that determines outcome of antiviral therapy. Baseline platelet count predicts the outcome of antiviral therapy in CT allele patients.

INTRODUCTION
Investigations on genetic determinants of chronic he patitis C (CHC) established that a single nucleotide polymorphism (SNP) in the interleukin (IL)28B gene promoter region affected the spontaneous and induced clearance of hepatitis C virus (HCV) [15] . Among 500 000 genetic variants which were analyzed genomewide, a few associated with the virologic response were identi fied, and showed variable frequency and importance across human ethnic groups [3,57] . The mechanism by which SNPs influence the outcome of HCV infection and its treatment is not clear. It is suggested that regula tion of the promoter region of IL28B in antiviral activity may also affect two other genes belonging to interferon (IFN)λ family encoded in this region [6,812] . According to some authors, IL28B SNPs are merely indicators of more complex mechanisms of CHC pathogenesis [13] . Some factors which determine the efficacy of antiviral therapy influence the course of CHC [14] . The impact of SNPs within the IL28B region on spontaneous and in duced HCV clearance suggests the possibility that poly morphism affects the chronic phase of hepatitis. On the basis of previous research, there is no clear evidence that there is a correlation between IL28B SNPs and progres sion of untreated CHC. In a metaanalysis by Romero Gomez et al [15] , an effect of IL28B polymorphism on the progression of fibrosis was disputed. Research evaluating liver cirrhosis and fibrosis progression after liver transplantation in HCVinfected patients indicated a connection with IL28B SNPs [16,17] . However, about 50% of patients with a sustained virological response (SVR) do not carry the favorable IL28B alleles [15] . The factors which increase the chance of a therapeutic response in these patients are not yet known. A detailed analysis of the course of therapy of CHC with pegylated IFNα and ribavirin (RBV) in the presence of a hazardous IL28B allele might better de lineate the clinical characteristics of this difficult-to-treat group of patients.
The aim of this study was to determine the impact of IL28B polymorphism on the progression of CHC, in order to identify predictors of effective therapy with IFN and RBV in the group of patients with unfavorable IL28B polymorphism.

MATERIALS AND METHODS
Sixtyfour patients of European origin (Caucasians) with CHC due to HCV genotype 1 were included in the pres ent study. Patients were treated with standard antiviral therapy with pegylated IFN 2a or 2b and RBV according to standard criteria of inclusion and exclusion. A liver biopsy was performed before treatment, and HCV viral load (COBAS ® TaqMan ® HCV 2.0) and insulin resistance index (HOMAIR: homeostasis model of assessment) was determined.
The histopathological assessment of necroinflam matory grade and fibrosis stage were scored using the METAVIR system, and the presence of fatty liver disease was taken into consideration. The course of therapy was monitored on the basis of early virologic response (EVR), end of treatment response (ETR) and SVR.
The presence of the IL28Brelated SNP rs 12979860 variant was determined in whole blood samples. Ge nomic DNA was isolated from peripheral blood samples using proteinase K ion exchange column extraction kit (A&A Biotechnology, Gdynia, Poland). An amplification product of 190 bp length was obtained using standard polymerase chain reaction with the following primers: 5' GCC TCT TCC TCC TGC GGG ACA AG and 5' GCG CGG AGT GCA ATT CAA CCC T. Following digestion with Bsh1236I (BstUI) restriction endonuclease, products were separated using agarose gel electropho resis. The variant C was digested by the enzyme into fragments. All laboratory procedures were performed on blinded samples.
Patient age, sex, measurable anthropometric param eters such as waist, waist/hip ratio, body mass index (BMI) and percentage of body fat from MALTRON ® measure ments were analyzed in the present study.
Based on estimated duration of infection, the fibrosis progression rate was defined as the ratio between fibrosis stage and HCV duration. The serum aminotransferase levels prior to the start of treatment and after 72 wk was assessed. Patient characteristics are presented in Table 1.
The complete blood count at baseline and its changes during the course of therapy were determined. Thresh olds for drug dose reduction were established: white blood cells < 750 cells/µL platelets < 70 000/µL, and hemoglobin < 10 g/L. For each patient the dose of IFN and RBV used in the therapy was established and expressed as a percentage of the recommended dosage.
The significance of differences in the clinical parameters as well as anthropometric, biochemical, histological and virological features between patients with and without a SVR was determined based on analysis of variance KruskalWallis rank sum, MannWhitney U and χ 2 tests. A similar analysis was performed on patients only with the IL28B CT genotype. In patients with CC and TT genotypes, analysis of the diversity of the SVR was not performed because of the disproportionate distribution and small number of patients who did or did not achieve a SVR (Table 1).
Using the KruskalWallis rank sum test, we assessed variations in the investigated clinical features between pa tients with different IL28B polymorphisms. In the logistic regression model we evaluated the odds ratio (OR) and 95% confidence interval (CI) of SVR, depending on the degree of inflammatory-necrotic activity, fibrosis scores, platelet count, baseline viral load, insulin resistance index (IR index) and IL28B polymorphisms ( Table 2). The OR was determined against cases of high inflammatorynecrotic activity, above grade 1 in the METAVIR score, advanced fibrosis according to METAVIR F > 1, pres ence of steatosis, viral load > 1 × 10 6 IU/mL, alleles TT and CT, baseline platelet count < 200 × 10 9 /L and IR index > 2.5.
The study was performed according to ethical guide lines of the 1975 Declaration of Helsinki. All patients were informed about the study, and provided written in formed consent.

RESULTS
In the MannWhitney U test, differences were found in inflammatorynecrotic activity (P = 0.011), stage of fi brosis (P = 0.009), fibrosis progression rate (P = 0.015), EVR and ETR (P = 0.00001), baseline viral load (P = 0.09), reduction in viral load at 3 mo of therapy (P = 0.0004) and the presence of a biochemical response after completion of therapy (P = 0.03) between the patients who achieved a SVR and those who did not. In the group of patients who achieved a SVR, a lower incidence of liver steatosis was observed significantly (χ 2 test, P = 0.062). In the χ 2 test, there were no significant differences between patients with and without SVR as regards sex, age, comorbidities, and anthropometric factors. Differ ences in the parameters mentioned above were not ob served among the three groups selected on the basis of the IL28B polymorphism. The χ 2 test showed differences in the rate of achievement of EVR (P = 0.02), ETR (P = 0.069) and SVR (P = 0.001) between groups of patients with CC, CT and TT alleles. The KruskalWallis rank sum test showed differences in the size of the reduction of viral load after 3 mo of therapy (P = 0.019) between groups of patients with CC, CT and TT alleles. The OR revealed that patients with the CC allele had a 25fold greater chance of achieving a SVR compared with those with the TT allele (P < 0.001) and a 5.4fold higher chance compared with those with the CT allele (P = 0.018) ( Table 2). Patients with low liver inflammation had a 3.9 times greater probability of achieving a SVR (P = 0.026), and patients with no or little fibrosis had 5.9 times greater probability (P = 0.008). HCV viral load < 1 × 10 6 IU/mL increased the chance of achieving a SVR by 3fold (P = 0.038), and an IR index < 2.5 increased the chance by 3.9fold (P = 0.029). The KruskalWallis rank sum test showed no differences in the IR index between groups of patients with CC, CT and TT alleles (P = 0.12). Spear man's rank correlation coefficient between IR index and stage of fibrosis and BMI was r = 0.618 and r = 0.605, respectively (P < 0.001). In the MannWhitney U test, differences were found in the IR index (P = 0.005) be tween patients with and without steatosis. The Kruskal Wallis rank sum test revealed borderline significance (P = 0.064) of increased fibrosis in patients with the TT allele ( Figure 1).
The MannWhitney U test established that patients who did not achieve a SVR had low platelet levels at baseline (P = 0.001) compared with subjects with a SVR. A similar trend was also found for neutrophils (P > 0.05). Patients without SVR bearing the CT allele, were charac terized by lower baseline platelet count (P = 0.003) and decreased (P = 0.02) drop of platelet count upon treat ment. Decreased platelet levels in patients with the CT al lele and absence of a SVR were accompanied by a higher incidence of requiring a dose reduction of IFN (P = 0.07). No differences in IFN and RBV dosage in patients with the CT allele or other allelic variants were found between groups with or without a SVR.

DISCUSSION
The present study observed an impact of IL28B SNPs by which both factors can influence the course and thera py of CHC.
In the present study 60% of patients with SVR had TT (8%) or CT (52%) genotypes, which are not known factors associated with higher IFN responsiveness. These values are similar to those described in previous reports [15] . Analysis of the differences in the course of therapy in patients with the CT allele and a SVR revealed higher baseline platelet and neutrophil levels. These fac tors were markedly reduced in the group of patients with therapeutic failure. The present results are consistent with existing data, where the platelet level was a predic tor of a rapid virological response [28] . In this study, a low baseline platelet count was significantly associated with the need for IFN dose reduction in the group without a SVR. In the group of patients with the CT allele, the platelet count was the only factor differentiating patients with and without a SVR. The chance of a SVR was 8fold higher in patients with the CT allele and higher platelet count compared with patients with low levels of platelets.
The platelet level is one of the factors used to esti mate the risk of the presence of advanced liver fibro sis [29] . In the present study, the platelet level also correlat ed with the stage of liver fibrosis (Figure 2). More severe fibrosis, and a lower baseline platelet level increased the risk of requiring a reduction in IFN dose and subsequent reduction in the chance of achieving a SVR [13] . Assess ment of the platelet level and the IL28B polymorphism can complement the decisionmaking algorithm for a pa tient's eligibility for antiviral therapy.
Differentiation of SNPs in the IL28B promoter re gion is the strongest factor determining the effectiveness of therapy with IFN and RBV. Despite the greater sever ity of fibrosis in patients with the TT genotype, there was no additional evidence of an IL28B SNP associated with progression of CHC. In patients with the IL28B CT allele, a low baseline platelet count is associated with a requirement to reduce IFN and a decreased chance of obtaining a SVR. on the efficacy of IFN and RBV in CHC, which was consistent with the results from previous research. In pa tients with TT and CT genotypes, greater severity of liver fibrosis and necroinflammation, presence of liver steato sis, higher HCV viral load, and lower baseline platelets, had a reduced chance of therapeutic success. In the OR of the SVR, the greatest differences concerned the IL28B polymorphism between CC and TT alleles. The differ ence was more than 4-fold for fibrosis in the comparison of the CC allele with the CT allele. For liver steatosis, low platelet level, necroinflammatory grade, IR index and ini tial viral load of HCV, the difference was 58fold ( Table  2). The results confirm that polymorphism in the IL28B is significantly related to the outcome of antiviral therapy of CHC.
The mechanism by which genetic factors influence the progression of fibrosis in CHC remains unclear [1821] . In the present study, patients with the TT genotype had a more active state of necroinflammation in the histo logical analysis, and borderline significance (P = 0.06) of greater severity of fibrosis ( Figure 1). This observation is consistent with previous results describing a higher inci dence of TT or CT allele in cases of cirrhosis and faster fibrosis progression in HCV-infected liver transplant re cipients and liver from donors with the TT genotype [16,17] . Taking into consideration that the correlation between the degree of fibrosis and IL28B was not statistically significant and there was no association with fibrosis pro gression rate, it was not possible to confirm the impact of the genotype on the progress of CHC. Thus, IL28B polymorphism is probably not the factor which directly affects the outcome of untreated CHC.
The correlation between insulin resistance and stage of fibrosis, presence of the liver steatosis, efficacy of IFN/RBV therapy and higher BMI observed in the pres ent study is consistent with the results from previous observations [2227] . No statistically significant correlation between insulin resistance and IL28B polymorphism was observed, indicating that there are different mechanisms

Background
Ineffective therapy for chronic viral hepatitis type C (CHC) determines the progression of liver disease to cirrhosis and primary hepatocellular carcinoma. Single nucleotide polymorphism (SNP) in the IL28B promoter gene region determines spontaneous and induced clearance of hepatitis C virus.

Research frontiers
Clinical aspects of the impact of IL28B SNPs on the efficacy of antiviral therapy and progression of CHC are not fully recognized. In the present study, potential factors influencing the efficacy of therapy in relation to IL28B polymorphism were investigated.

Innovations and breakthroughs
The results of the study confirm a significant role of SNPs in the IL28B gene among different predictive factors of a sustained virological response (SVR). The impact of IL28B SNPs on the SVR is not related to insulin resistance. In the case of the CT allele, a low baseline platelet count is associated with a reduced chance of a SVR. The phenomenon is connected with the stage of liver fibrosis and need for a reduction of interferon dose during therapy. The results suggest that the progression of CHC is faster in patients with the TT genotype.

Applications
Results of the study can complement the decision-making algorithm for eligibility of patients with CHC for antiviral therapy.

Terminology
Single nucleotide polymorphism is a variation of a single nucleotide usually found in non-coding microsatellite fragments of the genome. It may serve as the fingerprint for predicting susceptibility toward disease. IL28B SNPs include different variants localized in the gene promoter region, among which the most common in clinical practice is C/T SNP of rs 12979860.

Peer review
This study examined the association of SNPs of the IL28B, rs12979860 on progression of CHC. They found that SNPs of IL28B had any impact on CHC progression, in spite of apparently more advanced fibrosis among patients carrying TT genotype. In addition, they revealed that, in patients with the CT allele IL28B, low baseline platelets count is linked to the reduction of IFN and decreased chance of reaching the SVR. This study demonstrated a new characteristic of IL-28b in CHC.