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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Interleukin (IL)28B polymorphisms are associated with spontaneous clearance of hepatitis C virus (HCV) infection and response to therapy. Whether IL28B genotype affects fibrosis progression or clinical outcome is unclear. Our aim was to study the relationship between IL28B genotype and both histological and clinical outcomes in patients with chronic hepatitis C (CHC). Hepatic fibrosis was scored using the Ishak (0-6) scale; progression was defined as a 2-point increase in Ishak score between biopsies. Multiple logistic and Cox regressions were used to identify variables associated with fibrosis progression. In all, 1,483 patients were included in a baseline cross-sectional analysis, from which 276 were eligible for a paired biopsy analysis (median time between biopsies 4 years), and 400 for a clinical outcome analysis. At baseline biopsy, patients with IL28B CC genotype had significantly higher portal inflammation (2.4 versus 2.2) and alanine aminotransferase (ALT) levels (133 versus 105 U/L; P < 0.05 for all). In the paired biopsy analysis, there was no difference in the frequency of fibrosis progression between patients with IL28B CC and non-CC genotypes (17% versus 23%). In logistic regression, only higher baseline alkaline phosphatase, lower platelets, and greater hepatic steatosis were associated with fibrosis progression. Patients with IL28B CC were twice as likely to develop adverse clinical outcomes compared to non-CC (32% versus 16%; P = 0.007). Conclusion: IL28B CC genotype was associated with greater hepatic necroinflammation, higher ALT, and worse clinical outcomes in CHC patients. This suggests that IL28B CC is associated with a state of enhanced immunity that, on the one hand, can promote viral clearance, but alternately can increase necroinflammation and hepatic decompensation without enhancing fibrosis progression. (Hepatology 2013;58:1548–1557)

Abbreviations
ALT

alanine aminotransferase

AST

aspartate aminotransferase

BMI

body mass index

CHC

chronic hepatitis C

HAI

histological activity index

HALT-C

Hepatitis C Long-Term Treatment Against Cirrhosis

HCC

hepatocellular carcinoma

SVR

sustained virological response

Chronic hepatitis C (CHC) is a global health problem and can lead to cirrhosis, endstage liver disease and hepatocellular carcinoma (HCC).[1, 2] It is the most common cause of death from liver disease and indication for adult liver transplantation in the United States.[3] However, not all subjects with CHC will develop these serious sequelae; indeed, a majority of individuals will die with their disease rather than from their disease. Although several host, viral, and environmental factors have been linked with outcome of CHC,[4, 5] they do not completely explain the variable outcome of the disease. Recently, genome-wide association studies have identified several single nucleotide polymorphisms (SNPs), within and in the vicinity of three genes that encode interferon-lambda (IFN-λ).[6-10] The CC genotype of rs12979860 was strongly associated with resolution of HCV infection following treatment with peginterferon and ribavirin and was independent of race, with similar sustained virological response (SVR) rates among individuals of both European and African ancestry.[9] Moreover, rates of spontaneous and treatment-associated clearance of HCV infection for patients with the CC genotype were approximately double those for the TT genotype.[6, 9] These studies underscore the importance of the interleukin (IL)28B gene in the outcome of acute HCV infection and response to peginterferon-based therapy. However, the role of IL28B in the natural history of chronic HCV infection is not well understood.

A recent study suggested that the T allele of IL28B rs12979860 was more prevalent among patients with HCV-related cirrhosis compared to patients with mild CHC and that carriage of the T allele was associated with an increased risk of developing HCC.[11] Further, more severe necroinflammatory activity and fibrosis were found to be associated with homozygosity for the major T allele of another IL28B SNP (rs809917) in patients with CHC from Japan.[12] In contrast, Marabita et al.[13] showed no relationship between IL28B genotype and severity of liver fibrosis. Moreover, none of the previous studies have examined the relationship between the IL28B genotype and disease outcome as assessed by fibrosis progression using serial liver biopsies and hard clinical outcomes. Therefore, the primary aims of the current study were to investigate whether the previously identified IL28B SNP rs12979860 (CC, CT, or TT genotype) was associated with histological progression on serial liver biopsies in a large cohort of patients with CHC and to assess if there was any association of IL28B SNP rs12979860 with clinical outcomes.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information
Patients

Adult patients (age 18 or above) were analyzed from two cohorts: (1) patients participating in a long-term natural history study of CHC conducted at the Clinical Center of the National Institutes of Health (NIH)[14] including patients who were referred for evaluation and possible therapy who elected not to undergo treatment[14] (NIH Cohort); and (2) the Hepatitis C Long-Term Treatment Against Cirrhosis (HALT-C) Trial (HALT-C Cohort).[15] Patients in the NIH cohort underwent either a protocol liver biopsy or a recommended standard of care biopsy approximately every 5 years and were never treated before or between biopsies. The design of the HALT-C Trial has been described previously.[15] Briefly, patients with CHC who had failed to achieve an SVR after treatment with interferon with or without ribavirin and who had advanced fibrosis on liver biopsy (Ishak fibrosis score >3), with no history of hepatic decompensation or HCC were treated with peginterferon alfa-2a and ribavirin for 6 months (the lead-in phase of the trial). Patients who remained viremic during the lead-in phase of treatment (lead-in patients), those who experienced virological breakthrough or relapse after initial response (breakthrough/relapser patients) and those who were nonresponders to peginterferon and ribavirin outside of the HALT-C trial (express patients) were randomized to maintenance therapy (peginterferon alfa-2a 90 μg weekly) or to remain as untreated controls for the next 3.5 years. Liver biopsy was performed within 12 months prior to entry into the trial and then at 2 and 4 years following enrollment. Hepatic necroinflammation was scored using the histology activity index (HAI) scale (0-18) and hepatic fibrosis using the Ishak scoring system (0-6).[16] In the HALT-C trial hepatic steatosis was graded as 0 (<1%), 1 (1%-5%), 2 (5%-33%), 3 (33%-67%), and 4 (>67%) according to the percentage of hepatocytes with fat. In the NIH cohort, hepatic steatosis was graded on a 6-point scale as none, <5%, 5% to 25%, 26% to 50%, 51% to 75%, and 76% to 100% based on the proportion of hepatocytes with fat. For this analysis, the scoring scales were merged to none (<1% / 0%), trace (1%-5% / trace), mild (6%-33% / trace to 25%), and moderate to severe (all other categories).

All biopsies from the NIH cohort were read and scored by an expert hepatopathologist; biopsies from the HALT-C cohort were read and scored by a panel of 10 hepatopathologists, one of whom included the NIH hepatopathologist. Alcohol use was categorized as none, social drinking ≤3 drinks per day, and heavy alcohol consumption as >3 drinks per day. Patients were excluded if they had another cause for liver disease or if essential clinical or laboratory data were missing. None of the patients included in this analysis experienced an SVR.

Rationale for Combining the NIH and HALT-C Cohorts

The primary purpose for combining the two cohorts was to ensure representation of all stages of fibrosis in the analysis. We acknowledge the fact that the two cohorts are inherently different: the NIH cohort was untreated and had less severe fibrosis compared to the HALT-C cohort, while the HALT-C had all failed treatment previously and had advanced fibrosis. To control for this difference, all analyses were performed with the cohorts combined as well as separately to determine if the results were similar (Supporting Table S1).

Primary Outcome Analyses

Three primary outcome analyses were conducted: (1) cross-sectional analysis: IL28B genotypes were compared with liver biopsy grading and staging using the initial liver biopsy from the NIH cohort (n = 246) and the entry biopsy for the HALT-C cohort (n = 1,237). (2) Longitudinal analysis: to determine whether IL28B genotype was associated with fibrosis progression, we correlated the change in fibrosis score between biopsies with IL28B genotype. For this analysis we included only patients with paired liver biopsies a minimum of 1 year apart (and no more than 10 years apart) and absence of cirrhosis on the baseline biopsy since these patients could not progress. Patients from the HALT-C cohort who were randomized to receive low-dose peginterferon alfa-2a between liver biopsies were also excluded from this analysis as were breakthrough/relapsers because they received therapy beyond the lead-in phase of the trial (24 weeks of peginterferon alfa-2a and ribavirin). Based on these parameters, 168 patients from the NIH cohort were excluded: 161 because they did not have two biopsies within the required time period and seven with cirrhosis at entry (Fig. 1). In all, 1,039 patients from the HALT-C cohort were excluded: 306 who were not randomized, 134 randomized breakthrough/relapsers, 397 randomized to long-term peginterferon alfa-2a, 66 with no follow-up liver biopsy, and 136 with cirrhosis on entry biopsy (Fig. 1). Thus, data from a total of 276 patients were included, 78 from the NIH cohort and 198 from the HALT-C cohort. The 78 patients in the NIH cohort were never treated and 30 express patients in the HALT-C cohort received no treatment between biopsies. The remaining HALT-C patients were treated for 24 weeks after the first biopsy. The median duration between biopsies was 4 years (range 1.7 to 9.9 years). To limit the issue of biopsy sampling error, fibrosis progression was defined as a 2-point increase in Ishak score between the paired biopsies. Patients were divided into two groups: (1) patients with no fibrosis progression, defined as difference in the Ishak score of <2 between the biopsies; (2) patients with fibrosis progression, defined as 2 or greater increase in the Ishak score between biopsies. (3) Clinical outcomes analysis: For this analysis, only subjects from the control arm of HALT-C cohort (n = 400) were included because data on the clinical outcomes were prospectively collected over 3.85 years and adjudicated by a panel of three principal investigators using stringent criteria to confirm that a clinical event had indeed occurred. A clinical outcome was defined as one of the following: death, development of ascites, spontaneous bacterial peritonitis, variceal hemorrhage, hepatic encephalopathy, HCC, and increase in Child-Pugh-Turcotte score by 2 or more points on two consecutive clinic visits 12 weeks apart.

image

Figure 1. Derivation of the three analyses cohorts: for the baseline analysis there were 1,382 subjects enrolled in the HALT-C trial and 309 followed at NIH; 145 subjects from the HALT-C cohort were excluded either for missing IL28B results (n = 1430 or missing histology data (n = 2), and 63 subjects from the NIH cohort were excluded either for missing IL28B results (n = 58) or missing histology data (n = 5), leaving 1,483 for analysis. *Clinical outcome cohort: Only subjects randomized to the control arm of the HALT-C trial because data on the clinical outcomes were prospectively collected over 3.85 years. This was not done for the NIH cohort. **Paired biopsy cohort: For this analysis we included only patients with paired liver biopsies a minimum of 1 year apart (and no more than 10 years apart) and absence of cirrhosis on the baseline biopsy.

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Both studies were approved by the Institutional Review Board of the NIDDK, NIH and both cohorts signed a separate consent form for genetic testing.

IL28B SNP Analysis

Genotyping of the rs12979860 SNP was performed on all patients from the HALT-C and NIH cohorts with available DNA samples and who provided genetic consent as described[17] (Supporting Material).

Statistical Analysis

Baseline clinical characteristics and laboratory values of these patients and their relationships to fibrosis were examined. Variables analyzed included demographic factors including age, sex, race, and ethnicity, anthropometric indices (body mass index [BMI]), duration of infection, presence of diabetes, and alcohol consumption. The following laboratory and histological tests were included: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, alkaline phosphatase, total bilirubin, albumin, prothrombin time, platelet count, ferritin, and hepatic steatosis. Baseline variables were compared using chi-square, t test, or analysis of variance. Logistic regression was used to calculate odds ratios for the relationship between fibrosis progression and IL28B (CC versus CT or TT). Analyses of the combined cohorts included a variable indicating cohort (NIH or HALT-C). Other predictors of fibrosis progression were evaluated and those significant after backward selection were also included in the model. Change in fibrosis, HAI, and ALT were analyzed using an analysis of variance controlling for baseline levels. Clinical outcome rates were estimated using Kaplan-Meier estimates and significance was tested using the log-rank test and Cox proportional hazards regression. Analyses were conducted by cohort and with both cohorts combined. Data are presented as percent or mean and SD unless otherwise noted. SAS (Statistical Analysis Software, Cary, NC) v. 9.2 was used for statistical analyses.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Patient Demographics and Baseline Characteristics

A total of 309 patients were followed in NIH natural history studies and 1,382 patients were enrolled into the HALT-C trial. Sixty-three patients were excluded from the NIH cohort because of missing IL28B data in 58 and missing histology data in 5 patients. In all, 145 patients were excluded from the HALT-C trial because of missing IL28B data in 143 and missing histology data in 2 patients (Fig. 1). Thus, a total of 1,483 patients were available for the cross-sectional analysis, of whom 246 were from the NIH cohort and 1,237 from the HALT-C cohort.

The baseline demographic, laboratory, and histologic data are shown in Table 1. Overall, the mean age was 49 years, 69% were male, 75% were white, and the mean duration of infection was 28 years. In total, 21% of subjects had diabetes, 19% were teetotalers, and 25% had heavy alcohol consumption. The mean BMI was 30 and 86% were infected with HCV genotype 1. At baseline, the mean serum ALT was 112 U/L, mean serum AST was 87 U/L, and mean serum albumin, total bilirubin, and platelet count were within normal limits (Table 1). The distribution of IL28B genotypes was as follows: 25% of patients had CC genotype, 53% CT, and 22% TT. The mean Ishak fibrosis and HAI scores were 3.8 and 7.7, respectively; one-third of patients had cirrhosis at baseline, hepatic steatosis was absent or mild in 60%, and 9% had moderate-severe steatosis. No subject had concomitant nonalcoholic steatohepatitis.

Table 1. Baseline Characteristics of the NIH and HALT-C Studies
VariableNIHHALT-COverallP Valueb NIH vs. HALT-C
  1. a

    Mean, SD.

  2. b

    P values from analysis of variance or chi-square test (not adjusting for study).

  3. c

    First grouping was used by HALT-C, grouping in parentheses was used by NIH.

N patients2461,2371,483NA
Agea45.4, 9.049.9, 7.149.2, 7.7<0.0001
Male, %57.671.869.4<0.0001
Race   <0.0001
White, %76.674.374.7 
African American, %16.415.415.5 
Hispanic, %0.08.36.9 
Other, %7.02.12.9 
Duration of infection, yearsa18, 928, 827.9<0.0001
Diabetes, %1.123.820.9<0.0001
Alcohol history   <0.0001
None, %39.216.518.6 
Social, %29.659.656.8 
Heavy, %31.223.924.6 
BMIa27.7, 5.429.8, 5.529.5, 5.5<0.0001
IL28B36.222.725.0<0.0001
CC, %43.154.552.6 
CT, %20.722.822.4 
TT, %    
HCV genotype 1, %68.388.885.5<0.0001
ALTa118, 111111, 79112, 840.34
ASTa77, 7588, 6187, 630.045
Albumina4.2, 0.343.9, 0.403.9, 0.40<0.0001
Alkaline phosphatasea79, 2599, 44.096, 43<0.0001
Plateletsa208, 61167, 65173, 66<0.0001
Total bilirubina0.74, 0.330.79, 0.420.78, 0.410.086
Serum ferritina169, 199354, 391338, 382<0.0001
Log serum ferritina2.0, 0.412.3, 0.462.3, 0.46<0.0001
Fibrosis   <0.0001
0–2, %56.98.316.3 
3–4, %33.353.149.8 
5–6, %9.838.633.9 
Fibrosisa2.3, 1.74.1, 1.33.8, 1.5<0.0001
HAIa8.6, 2.87. 6, 2.17.7, 2.3<0.0001
Lobular inflammationa3.3, 1.02.6, 0.92.7, 1.0<0.0001
Portal inflammationa2.2, 1.22.2, 0.82.2, 0.80.76
Periportal inflammationa3.0, 1.62.8, 1.02.8, 1.10.029
Steatosisc   <0.0001
None40.218.422.0 
Trace15.942.037.6 
Mild36.630.431.4 
Moderate to severe7.39.39.0 
Steatosisa1.1, 1.01.3, 0.91.3, 0.90.005

Patients in the HALT-C trial were more likely to be older (50 versus 45 years), male (72% versus 58%), of Hispanic race (8.3% versus 0%), have longer duration of infection (29 versus 19 years), have a higher BMI (30 versus 28), a higher prevalence of diabetes (24% versus 1%), more likely to consume alcohol, and be infected with HCV genotype 1 (89% versus 68%) compared to the NIH cohort. Serum AST (but not ALT), alkaline phosphatase, and ferritin were higher and serum albumin and platelet counts were lower in the HALT-C cohort as compared to the NIH cohort. The HALT-C cohort had a lower frequency patients with IL28 genotype CC compared to the NIH cohort (22% versus 36%; P < 0.0001). The NIH patients had a higher mean HAI score (8.6 versus 7.6; P < 0.0001), higher lobular and periportal inflammatory scores, and a greater proportion of subjects with no hepatic steatosis compared to the HALT-C cohort but the mean fibrosis score was significantly higher in the HALT-C cohort compared to the NIH cohort (4.1 versus 2.3; P < 0.0001). Fifty-seven percent of the NIH cohort had mild disease, Ishak fibrosis scores ranging between 0-2, compared to only 8% of HALT-C patients. In contrast, 39% of the HALT-C cohort had cirrhosis (Ishak 5-6) compared to 10% of the NIH cohort.

Cross-Sectional Analysis

Association of Baseline Clinical and Laboratory Factors With IL28B Genotype

White patients had a higher frequency of IL28b genotypes CC and CT compared to TT (79% and 79% versus 61%, respectively; P < 0.0001; Table 2). Conversely, African-American patients had a higher frequency of IL28B genotype TT compared to CC and CT (31% versus 6% and 14%, respectively; P < 0.001). Interestingly, patients with the IL28B CC genotype were less likely to be infected with HCV genotype 1 than the CT and TT genotypes (76% versus 87% versus 93%, respectively; P < 0.0001; Table 2, Fig. 2). Serum ALT, a surrogate marker of inflammation, was higher in patients with the CC genotype compared to patients with non-CC genotype.

image

Figure 2. Frequency of IL28B genotypes by HCV viral genotype (cross-sectional analysis): 1,483 patients were included in the cross-sectional analysis.

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Table 2. IL28B Genotype With Clinical, Laboratory and Histological Characteristics n = 1483 (NIH and HALT-C Cohorts Combined)
VariableCCCTTTP Valueb 3 GroupsP Valueb CC vs. not CC
  1. a

    Mean, SD.

  2. b

    P values from analysis of variance or chi-square test (not adjusting for study).

  3. c

    First grouping was used by HALT-C, grouping in parentheses was used by NIH.

N patients370780333NANA
Agea49.5, 7.448.9, 7.649.5, 8.20.280.33
Male, %72.2%70.5%64.0%0.0410.19
Race   <0.0001<0.0001
White, %78.978.760.7  
African American, %6.013.630.6  
Hispanic, %7.06.47.8  
Other, %8.11.30.9  
Duration of infection, yearsa27.1, 9.227.0, 8.426.5, 8.90.710.68
Diabetes, %17.322.820.80.110.057
Alcohol history   0.530.59
None, %17.317.921.6  
Social, %56.357.555.7  
Heavy, %26.424.722.6  
BMIa29.3, 5.629.8, 5.529.3, 5.40.280.31
HCV genotype   <0.0001<0.0001
1, % (n = 1263)76.486.992.5  
2, % (n = 97)12.05.72.7  
3, % (n = 91)10.15.53.3  
Other, % (n = 26)1.61.91.5  
ALTa133, 113107, 73100, 66<0.0001<0.0001
ASTa93, 8384, 54886, 560.0680.065
Albumina4, 0.43.9, 0.43.9, 0.40.00450.017
Alkaline phosphatasea94, 4497, 4398, 410.430.25
Plateletsa171, 65174, 66172, 670.820.67
Total bilirubina0.7, 0.40.8, 0.40.8, 0.50.180.38
Serum ferritina257, 270354, 409384, 404<0.0001<0.0001
Log serum ferritina2.2, 0.42.3, 0.52.4, 0.5<0.0001<0.0001
Fibrosis   0.0760.046
0–2, %20.315.813.2  
3–4, %48.948.753.5  
5–6, %30.835.533.3  
Fibrosisa3.6, 1.63.8, 1.53.8, 1.50.0700.021
HAIa8.0, 2.57.7, 2.27.6, 2.20.0150.0069
Lobular inflammationa2.7, 1.02.7, 0.92.8, 0.90.430.84
Portal inflammationa2.4, 0.92.2, 0.82.1, 0.8<0.0001<0.0001
Periportal inflammationa2.9, 1.22.8, 1.12.7, 1.00.0600.025
Steatosisc   <0.0001<0.0001
None34.318.117.4  
Trace34.139.437.5  
Mild24.932.137.2  
Moderate to severe6.810.57.8  
Steatosisa1.0, 0.91.4, 0.91.4, 0.9<0.0001<0.0001
Association of IL28B With Histological Severity

On cross-sectional analysis, subjects with IL28B CC genotype had lower Ishak fibrosis scores compared to those with IL28B CT and TT genotypes combined (3.6 versus 3.8; P = 0.021), but this was not significant when all three groups were compared (3.60 versus 3.80 versus 3.81; P = 0.07; Table 2 and Supporting Fig. 1) or when the cohorts were analyzed separately (Supporting Table S1). Subjects with IL28B CC genotype had higher total HAI scores as well as periportal and portal necroinflammation compared to non-CC genotypes (Table 2). Subjects with IL28B CC genotype had milder hepatic steatosis compared to CT and TT (1.0 versus, 1.4 and 1.4, respectively; P < 0.0001) and were more likely to have no hepatic steatosis. Similar results were obtained when the two cohorts were analyzed separately (Supporting Table S1).

Longitudinal Analysis

This analysis examined the association between IL28B genotype with fibrosis progression based on paired biopsies from the untreated NIH cohort (n = 78) and the control arm of the HALT-C trial (n = 198). Baseline characteristics of the two cohorts were different in several aspects (Supporting Tables S2 and S3). Compared to the HALT-C cohort, subjects in the NIH cohort were younger, more likely to be female, white, have a shorter duration of infection, less likely to have diabetes or consume alcohol, and have laboratory and histological features consistent with the presence of milder liver disease (Supporting Table S2). The IL28B CC genotype was twice as frequent in the NIH cohort compared to the HALT-C cohort (26% versus 13%, respectively; P = 0.02). Overall, the distribution of IL28B genotypes was 17% CC, 54% CT, and 30% TT.

Association of IL28B Genotype With Histological Progression

Overall, a 2-point increase in Ishak fibrosis score was observed in 60/276 (22%; Table 3a). Progression of fibrosis occurred more frequently in the HALT-C cohort compared to the NIH cohort, P = 0.0037. There was no difference in the frequency of fibrosis progression between patients with IL28B genotype CC (17%) and non-CC (23%), both in unadjusted analysis and after adjusting for baseline platelets, alkaline phosphatase, and hepatic steatosis (P = 0.51). The mean change in Ishak fibrosis scores was 0.41 among patients with IL28B CC genotype and 0.51 among those with IL28B CT or TT genotype (P = 0.95; Table 4, Supporting Fig. 2). Results were similar when the cohorts were analyzed separately, HALT-C (0.46 versus 0.58, P = 0.70) and NIH cohorts (0.35 versus 0.33, P = 0.60; Table 4).

Table 3. Fibrosis Progression (2-Point Increase in Ishak Score) and Clinical Outcomes by IL28B Genotype and Cohort
Table 3a
 nFibrosis Progression n (%)Odds Ratio (95% CI)P Value
  1. a

    Odds ratios and P values adjusted for cohort and baseline platelets, AP, and steatosis using logistic regression.

  2. b

    Odds ratios and P values adjusted for baseline platelets, AP, and steatosis using logistic regression.

  3. c

    Hazard ratio and P value adjusted for baseline diabetes, cirrhosis, albumin, and total bilirubin using Cox regression.

All
CC468 (17%)Unadjusted: 0.85 (0.37–1.981)0.71
CT/TT23052 (23%)Adjusted:a 0.72 (0.28–1.89)0.51
NIH
CC202 (10%)Unadjusted: 0.96 (0.18–5.21)0.97
Adjusted:b 0.38 (0.04–3.70)0.40
CT/TT586 (10%)
HALT-C
CC266 (23%)Unadjusted: 0.82 (0.31–2.17)0.69
CT/TT17246 (27%)Adjusted:b 0.89 (0.30–2.62)0.83
Table 3b
 nFibrosis Progression n (%)Odds Ratio (95% CI)P Value
HALT-C
CC3416 (32%)Unadjusted: 2.33 (1.34–4.06)0.0029
CT/TT29456 (16%)Adjusted:c 2.30 (1.31–4.03)0.0037
Table 4. Change in Mean Fibrosis, HAI Scores, and Serum ALT Level Between Biopsies by IL28B Genotype and Cohort
 VariableaP Value StudybP Value IL28BbP Value Interactionb
  1. a

    Mean, SD.

  2. b

    P values are from an analysis of variance with factors for study, IL28B (CC vs. CT and TT), study and IL28B interaction, and fibrosis, HAI, or ALT at baseline.

Ishak fibrosis score
ALL
CC (n = 46)0.41, 1.260.080.950.55
CT/TT (n = 230)0.51, 1.25
NIH
CC (n = 20)0.35, 1.04NA0.60NA
CT/TT (n = 58)0.33, 0.87
HALT-C
CC (n = 26)0.46, 1.42NA0.70NA
CT/TT (n = 172)0.58, 1.35
HAI score
ALL
CC (n = 46)−0.13, 2.910.00690.530.060
CT/TT (n = 230)0.49, 2.74
NIH
CC (n = 20)0.10, 2.99NA0.40NA
CT/TT (n = 58)0.71, 3.18
HALT-C
CC (n = 26)−0.31, 2.90NA0.30NA
CT/TT (n = 172)0.42, 2.58
ALT U/L
ALL
CC (n = 46)−51.8, 105.10.790.190.37
CT/TT (n = 230)2.7, 75.9
NIH
CC (n = 20)−84.4, 125.8NA0.35NA
CT/TT (n = 58)11.8, 114.4
HALT-C
CC (n = 26)−33.6, 89.2NA0.64NA
CT/TT (n = 172)−0.1, 59.8
Association of IL28B Genotype With Change in HAI Score and Serum ALT Level

We also explored the relationship between change in HAI scores and serum ALT level between liver biopsies with IL28B genotype. At baseline, patients with IL28B genotype CC had higher total HAI scores as well as portal and periportal inflammatory scores. Serum ALT and AST levels were higher and log serum ferritin lower at baseline in patients with IL28B genotype CC compared to those with genotypes CT/TT (Supporting Table 2). HAI scores and serum ALT levels improved between biopsies in patients with IL28B CC genotype (mean change −0.13 and −52 U/L, respectively) compared to IL28B non-CC genotype (mean change 0.49 and 3 U/L, respectively) but these differences were not significant (Table 4, Supporting Figs. 3,4).

Factors Associated With Fibrosis Progression

In a logistic regression model to identify factors associated with a 2-point increase in Ishak fibrosis score, low platelet count, elevated alkaline phosphatase, and more severe hepatic steatosis at baseline liver biopsy were the best predictors of fibrosis progression. IL28B genotype non-CC versus CC was not significantly associated with fibrosis progression and addition of IL28B genotype to the model did not improve the fit.

Clinical Outcomes Analysis

To determine whether IL28B genotype was associated with clinical outcome, we restricted the analysis to the untreated HALT-C cohort who were prospectively observed every 3 months for 3.85 years for the development of clinical outcomes (death n = 7, ascites n = 7, spontaneous bacterial peritonitis n = 1, variceal hemorrhage n = 3, hepatic encephalopathy n = 6, HCC n = 11, and increase in Child-Turcotte-Pugh score by ≥2 points at two consecutive study visits n = 37, total events n = 72). There was no histological requirement for this analysis; thus, 400 subjects who were randomized to the control arm of HALT-C were analyzed and included 50 IL28B genotype CC and 350 IL28B genotype CT/TT subjects. Interestingly, untreated subjects with IL28 CC genotype were twice as likely to develop an adverse clinical outcome compared to subjects with IL28B non-CC genotype by life table analysis (32% versus 16%, respectively; P = 0.003; Table 3b and Fig. 3). This difference became apparent within the first 6 months of randomization and continued to increase over the period of follow-up. This finding remained significant after adjusting for the presence of diabetes at baseline, cirrhosis, albumin, and bilirubin levels (P = 0.004). The result was also independent of fibrosis stage and observed in those with baseline cirrhosis and bridging fibrosis on baseline biopsy.

image

Figure 3. Life table analysis of clinical outcomes by IL28B genotypes: Clinical outcomes were defined as death, ascites, spontaneous bacterial peritonitis, variceal hemorrhage, hepatic encephalopathy, HCC, and increase in Child-Turcotte-Pugh score by ≥2 points at two consecutive study visits in 400 untreated HALT-C subjects prospectively observed for 3.8 years.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Several recent studies that have examined the association between IL28B genotype and disease severity (hepatic fibrosis and necroinflammation) in patients with CHC have yielded contradictory results.[11-13, 18-20] Some studies have shown an association between IL28B rs12979860 genotype CC (or rs 809917 TT) with more advanced fibrosis or cirrhosis,[12, 20] and some have shown an association of the minor, IL28B rs12979860 genotype TT (or rs 809917 GG) with more advanced fibrosis or cirrhosis,[11, 18, 19] while other studies have reported no association of IL28B genotype with fibrosis.[13] There may be multiple reasons for the discrepant results, including small sample size in some studies, different patient cohorts, sample bias, study design-cross-sectional versus longitudinal, and imprecise estimates of fibrosis progression. Two studies determined a fibrosis progression rate by calculating the ratio of fibrosis stage to the estimated duration of infection.[13, 20] This method assumes that fibrosis progression occurs linearly over time. Using this approach, one study showed no association between IL28B genotype and fibrosis progression,[13] while the other suggested that the IL28B rs 809917 GG genotype was associated with a slower rate of fibrosis progression, particularly in patients with non-1 HCV genotype.[20]

In this analysis, when fibrosis progression was assessed using a stringent definition of a 2-point worsening in Ishak score between paired biopsies, we found no association between IL28B genotype with fibrosis progression after controlling for baseline platelet count, alkaline phosphatase, and hepatic steatosis. This result strongly suggests no association between IL28B genotype and fibrosis progression.

A significant finding of this analysis was the observation that HALT-C subjects with IL28B genotype CC, who received no treatment beyond the 24 week lead-in period, had twice the rate of adverse outcomes when compared to subjects with IL28B genotype non-CC. This finding was present even after controlling for baseline factors associated with a poor outcome. We speculate that prior nonresponders with the IL28B CC genotype may have had a worse outcome than nonresponders with IL28B non-CC genotypes due to a more vigorous immune response that was insufficient to result in viral clearance, but sufficient to cause greater liver cell injury as evidenced by greater hepatic necroinflammation and serum ALT levels. Two other studies have noted an association of greater necroinflammation and higher serum ALT levels in patients with IL28B genotype CC, but neither examined its relationship to clinical outcomes.[20, 21] It is also possible that other recently identified IL28B variants that are in linkage disequilibrium with IL28B CC genotype may account for the differences.[22, 23]

An apparent paradox in this study was that the higher indices of inflammation observed in subjects with IL28B genotype CC were associated with more severe clinical outcomes, but not with fibrosis progression. We offer several possible explanations. First, the duration between the paired biopsies (median 4 years) may not adequately capture the rate of fibrosis progression with a small sample size and slow fibrosis progression, resulting in type II error. Second, fibrosis progression, although important, might not be the only cause of adverse clinical outcomes in patients with CHC. Indeed, natural history studies have reported that a majority of patients with cirrhosis maintain stable liver disease without clinical decompensation for many years, suggesting that other factors likely contribute to the development of clinical events.[24-26] Third, production of selective proinflammatory, antiviral cytokines by cells of the innate immune response in patients with IL28B genotype CC, such as interferon gamma, could result in more hepatic inflammation and higher rates of viral clearance during early infection, but conversely precipitate clinical decompensation in subjects with late-stage disease even in the absence of fibrosis progression.[27] Fourth, it is possible that differences in baseline characteristics between the two cohorts may account for some of the associations found with IL28B. However, analyzing the cohorts separately yielded similar findings (Supporting Table S1). Finally, clinical outcomes are much better endpoints of disease progression than fibrosis staging, and therefore should be the more biologically relevant measure of phenotype-genotype correlation.

IL28B genotype CC was associated with a lower frequency of HCV genotype 1 infection and hepatic steatosis compared to IL28B genotypes CT or TT. These appear to be consistent findings among studies that have examined these relationships.[11, 28-30] The reason for the association with HCV genotype 1 is not clear but the findings suggest that either HCV non-1 genotypes preferentially infect subjects with IL28B genotype CC or that HCV genotype 1 infections may be more effectively cleared by those with IL28B CC genotype compared to non-1 HCV genotype. It is possible that prior therapy of the HALT-C cohort may have introduced bias into the analysis by enrichment of the HALT-C cohort (predominantly HCV genotype 1) with IL28B non-CC genotypes. However, when we examined the NIH cohort, all of whom were untreated, the same association with IL28B genotype and HCV genotype was found.

This study has several strengths that are worth highlighting. Patients were derived from two pedigreed cohorts that encompassed a wide spectrum of disease severity. Liver biopsies were evaluated by a panel of expert hepatopathologists in the majority of cases and a robust definition of fibrosis progression was used, a 2-point increase in fibrosis score, that should limit the effect of sampling error on liver biopsy. No previous study has used paired biopsies, which we believe is the best approach to explore the relationship between IL28B and disease progression. Subjects in the HALT-C trial were prospectively followed every 3 months for the development of clinical decompensation and all events were adjudicated by a panel of three rotating investigators before being entered into the trial database. A potential limitation was that all the subjects in the HALT-C cohort had received a prior course of therapy that may have influenced liver histology such that patients with IL28B genotype CC with milder disease may have responded to therapy, thereby enriching the cohort with subjects with more severe activity and fibrosis. However, since response to interferon-based therapy is independent of baseline ALT level[31] (a surrogate marker of HAI score), this would likely be true for other IL28B genotypes with milder activity and fibrosis. Moreover, the last course of interferon was completed a median of 15 months prior to the baseline biopsy in HALT-C and a minimum of 3.5 years prior to the follow-up biopsy. In addition, therapy was shown to have no influence on fibrosis progression in the HALT-C trial[15] and subjects with IL28B genotype CC from the untreated NIH cohort had greater inflammation than subjects with IL28B non-CC genotypes (Supporting Table S1).

In summary, we demonstrate that IL28B genotype was not associated with fibrosis progression in patients with CHC. However, the IL28B CC genotype was associated with greater hepatic necroinflammation, higher serum ALT levels, and a higher rate of clinical outcomes. This suggests that IL28B genotype CC may be associated with a state of enhanced antiviral immune response that promotes viral clearance and inflammation, but not fibrosis progression. This further suggests that there are mechanisms for fibrogenesis that are independent of inflammation.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

We thank Dr. Richard Chen for contributions, who passed away during revision of this work. We also thank the HALT-C investigators without whom this work could not have been done.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep26506-sup-0001-supptable.doc232K

Supporting Table S1: IL28B genotype with Clinical, Laboratory and Histological Characteristics, NIH (246) and HALT-C (1237) Cohorts Separately

Table S2: Clinical, Laboratory and Histological Characteristics of the Longitudinal Cohort at Baseline.

Table S3. Clinical, Laboratory and Histological Characteristics of the Longitudinal Cohort at Baseline by IL28B (NIH and HALT-C combined)

hep26506-sup-0002-suppfig1.tif3987KSupplementary figure 1: Frequency of IL28b genotypes by HCV viral genotype (Cross-sectional analysis): 1483 patients were included in the cross-sectional analysis.
hep26506-sup-0003-suppfig2.tif2457KSupplementary figure 2: Change in Ishak fibrosis score between paired biopsies by IL28b genotypes (longitudinal analysis): 276 patients were included in the longitudinal analysis.
hep26506-sup-0004-suppfig3.tif2352KSupplementary figure 3: Change in HAI score between the paired biopsies by IL28b genotypes (longitudinal analysis): 276 patients were included in the longitudinal analysis.
hep26506-sup-0005-suppfig4.tif2241KSupplementary figure 4: Change in ALT levels between the paired biopsies by IL28b genotypes (longitudinal analysis): 276 patients were included in the longitudinal analysis.

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