Complementary role of vitamin D deficiency and the interleukin-28B rs12979860 C/T polymorphism in predicting antiviral response in chronic hepatitis C

Authors

  • Davide Bitetto,

    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
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  • Giovanna Fattovich,

    1. Gastroenterology Clinic, Department of Medicine, Azienda Ospedaliero-Universitaria, University of Verona, Verona, Italy
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  • Carlo Fabris,

    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
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  • Elisa Ceriani,

    1. Department ofClinical and Experimental Medicine, University of Piemonte Orientale, Novara, Italy
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  • Edmondo Falleti,

    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
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  • Ezio Fornasiere,

    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
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  • Michela Pasino,

    1. Gastroenterology Clinic, Department of Medicine, Azienda Ospedaliero-Universitaria, University of Verona, Verona, Italy
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  • Donatella Ieluzzi,

    1. Gastroenterology Clinic, Department of Medicine, Azienda Ospedaliero-Universitaria, University of Verona, Verona, Italy
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  • Annarosa Cussigh,

    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
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  • Sara Cmet,

    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
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  • Mario Pirisi,

    1. Department ofClinical and Experimental Medicine, University of Piemonte Orientale, Novara, Italy
    2. Research Centre for Autoimmune Diseases, Novara, Italy
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  • Pierluigi Toniutto

    Corresponding author
    1. Department of Medicine and Pathology Clinical and Experimental, Medical Liver Transplantation Unit, Internal Medicine, University of Udine, Udine, Italy
    • Department of Medicine and Pathology Clinical and Experimental, Internal Medicine, Medical Liver Transplantation Unit, University of Udine, Udine, Italy
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    • fax: (39)-559490


  • Potential conflict of interest: Nothing to report.

  • Supported in part by grants from the Ricerca Sanitaria Finalizzata Program, Regione Piemonte, Italy (to M. P.).

Abstract

The widely accepted interleukin-28B (IL-28B) rs12979860 C/T polymorphism and the more recently proposed vitamin D serum concentration are two novel predictors of the response to antiviral treatment in chronic hepatitis C virus (HCV) infection. This study aimed to verify whether the IL-28B rs12979860 C/T polymorphism and pretreatment serum vitamin D levels have independent or complementary roles in predicting the rates of sustained viral response (SVR). The present study included 211 consecutive, treatment-naïve chronic HCV patients who had their pretreatment serum 25-OH vitamin D level and IL-28B rs12979860 C/T genotype determined. Overall, SVR was achieved by 134/211 (63.5%) patients and by 47/110 (42.7%) patients infected with difficult-to-treat HCV genotypes. On multivariate analysis, SVR was predicted by the HCV genotype, the IL-28B rs12979860 C/T polymorphism, and gamma-glutamyl transpeptidase, HCV RNA, cholesterol, and 25-OH vitamin D serum levels, with an area under the receiver operating characteristic (ROC) curve of 0.827. When difficult-to-treat HCV genotypes were analyzed separately, the SVR was predicted by the IL-28B rs12979860 C/T polymorphism, viral load, and serum vitamin D level, with an area under the ROC curve of 0.836. Moreover, by categorizing these latter patients into four groups—C/C homozygotes with vitamin D levels >20 ng/mL (group A) or ≤20 ng/mL (group B) and C/T heterozygotes or T/T homozygotes with vitamin D levels >20 ng/mL (group C) or ≤20 ng/mL (group D)—a significant linear trend was observed, with SVR rates in the following descending order: group A, 18/21 (85.7%); group B, 6/11 (54.5%); group C, 14/38 (36.8%); and group D, 9/40 (22.5%) (P < 0.0001). Conclusion: Vitamin D serum levels are complementary to the IL-28B rs12979860 C/T polymorphism in enhancing the correct prediction of the SVR in treatment-naïve chronic hepatitis C. (HEPATOLOGY 2011;)

Chronic hepatitis C affects 170 million people worldwide1 and is a major cause of chronic liver disease. Combination therapy with pegylated interferon (PEG-IFN) alpha and ribavirin is the current standard of care, but it has limited efficacy and a high cost. During the last decade, several modifiable and nonmodifiable parameters have been identified to help clinicians predict the probability of achieving a sustained viral response (SVR) prior to treatment in individual patients.2-7 Although new, specifically targeted antiviral drugs are on the horizon, they will not substitute for interferon (IFN)-based therapies in the near future, and they are expected to be more potent but also more expensive and toxic than the current standard of care. Therefore, refining the ability to predict response to current antiviral regimens remains an important research goal.

During the last year, two new predictors of response to antiviral treatment have emerged: the interleukin-28B (IL-28B) rs12979860 C/T polymorphism and vitamin D serum concentration. The IL-28B rs12979860 C/T polymorphism, located on chromosome 19 upstream of the gene encoding IFN-γ3, represents a host-related, nonmodifiable variable that strongly predicts the response to antiviral treatment. Among hepatitis C virus (HCV)-1–infected patients,8-10 SVR rates higher than 60%-80% were achieved by C/C homozygotes compared with the 15%-30% achieved by carriers of the T/T or T/C alleles.8, 11 Given the strength of this association, any new or old pre-treatment predictor of response must be compared against it. The second novel predictor, serum vitamin D concentration, is also of great interest because it is easily modifiable by dietary supplementation. Based on several recent reports demonstrating that vitamin D appears to possess important immunomediated and antiproliferative effects, Petta et al.12 investigated patients with genotype 1 chronic hepatitis C who underwent standard PEG-IFN plus ribavirin treatment and showed that the serum 25-OH vitamin D concentration was an independent predictor of viral clearance. Others have demonstrated that cholecalciferol supplementation added to combination therapy with PEG-IFN plus ribavirin could enhance the rates of SVR in patients with genotype 1 chronic hepatitis C.13 Finally, a retrospective analysis by our group involving a cohort of patients with recurrent hepatitis C after liver transplantation supports both above-mentioned observations14 (i.e., the prediction of lower SVR rates in the presence of vitamin D deficiency and the usefulness of vitamin D supplementation during antiviral treatment to promote a SVR). However, the role of the serum vitamin D concentration as a predictor of SVR has not been evaluated in conjunction with the IL-28B rs12979860 C/T polymorphism. Therefore, the aims of the present study were: (1) to ascertain whether vitamin D deficiency influences SVR rates in genotype 1–infected patients and those patients not infected with genotype 1 and (2) to verify whether the IL-28B rs12979860 C/T polymorphism and pretreatment serum vitamin D levels are independent or complementary predictors of treatment-induced viral clearance.

Abbreviation

cEVR, complete early viral response; CI, confidence interval; EOT, end of treatment viral response; HCV, hepatitis C virus; IFN, interferon; IL-28B, interleukin-28B; OR, odds ratio; PEG-IFN, pegylated interferon; ROC, receiver operating characteristic; RVR, rapid viral response; SVR, sustained viral response.

Patients and Methods

Patients.

The study population included a total of 211 consecutive, treatment-naïve hepatitis C patients of Caucasian ethnicity who received antiviral treatment at one of three academic centers in northern Italy (the Medical Liver Transplantation Unit at the University of Udine [n = 71; 33.6%], the Department of Gastroenterology at the University of Verona [n = 91; 43.1%], and the Department of Clinical and Experimental Medicine at the University of Piemonte Orientale [n = 49; 23.3%]) from September 2005 to October 2009. Chronic hepatitis C infection was defined by presence of anti–hepatitis C virus antibodies, serum HCV RNA positivity, and persistent elevation of alanine aminotransferase for at least 6 months. In addition, 144 patients had had a liver biopsy performed within the 6 months preceding the start of antiviral therapy. Exclusion criteria were decompensated liver cirrhosis (Child-Pugh score >6), presence of hepatocellular carcinoma (HCC), human immunodeficiency virus coinfection, hepatitis B virus coinfection, autoimmune liver disease defined according to validated diagnostic criteria,15 genetic liver disease (e.g., Wilson disease, hemochromatosis), concomitant use of drugs known to affect serum vitamin D concentration, and active intravenous drug use. The main clinical and demographic characteristics of the studied population are reported in Table 1. The study was conducted according to the principles of the Declaration of Helsinki and approved by the hospital institutional review boards and ethical committees. All patients gave written informed consent to participate in the study. The following variables were recorded prior to treatment: age, sex, body weight, height, and body mass index (kilograms per meters squared). Alcohol intake was evaluated by a questionnaire and quantified in grams per day. An overnight fasting blood sample was drawn to determine baseline blood tests, including HCV RNA quantification using real-time polymerase chain reaction (TaqMan, Roche), and HCV genotype using the InnoLipa genotyping kit (Innogenetics, Zwijndrecht, Belgium).

Table 1. Baseline Characteristics of the Study Population
  • Abbreviations: ALT, alanine aminotransferase, GGT, gamma-glutamyl transpeptidase; HCV, hepatitis C virus; HOMA, homeostasis model assessment,

  • Continuous variables are presented as the median (range); categorical variables are presented as frequency (%).

  • *

    Available in 144 patients.

Age, years47 (18-77)
Male sex109 (51.7%)
Body mass index, kg/m224 (16-39)
HCV genotype
 195 (45.0%)
 263 (29.9%)
 338 (18.0%)
 4-515 (7.1%)
HCV RNA, ×103 IU/mL700 (3.38-13,000)
Cholesterol, mg/dL170 (72-327)
ALT, IU/mL68 (12-428)
GGT, IU/mL38 (6-875)
HOMA2.0 (0.2-17.9)
Use of peginterferon alfa-2b149 (70.6%)
Grading pretreatment*2 (0-9)
Staging pretreatment*2 (0-6)
Alcohol consumption, g/day0 (0-50)

Vitamin D Assay and Homeostasis Model Assessment Score.

For all 211 patients, a serum sample, collected before starting antiviral therapy, was separated and stored at −80°C until used. This sample was available to measure pretreatment serum 25-OH vitamin D levels using a chemoluminescent immunoassay on a Liaison automatic analyzer (DiaSorin Inc., Stillwater, MN). The data are expressed as nanograms per milliliter. The same serum samples were also used to measure glucose and insulin serum levels. The insulin level was determined using an electro-chemiluminescence immunoassay (Insulina Immulite 2000, Medical System S.p.A., Genova, Italy). The homeostasis model assessment score was calculated as described16 using fasting glucose and insulin levels.

Molecular Biology.

Genotyping for the IL-28B rs12979860 C/T polymorphism was performed by polymerase chain reaction–based restriction fragment length polymorphism assay. Genomic DNA was extracted from whole blood samples using the QIAamp DNA blood mini kit (Qiagen, Milan, Italy) according to the manufacturer's instructions. A 242-bp product was obtained with the forward primer 5′-GCTTATCGCATACGGCTAGG-3′ and the reverse primer 5′-AGGCTCAGGGTCAATCACAG-3′, which were newly designed using the NCBI Primer-Blast Tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Polymerase chain reaction amplification was performed in a total volume of 10 μL containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, Tween-20 0.01%, 0.2 mM deoxyribonucleotides, 2-4 pmol of each primer, 2 mM MgCl2, and 0.5 units hot-start Taq DNA polymerase (RighTaq, Euroclone, Milan, Italy). Samples containing 10 ng of genomic DNA were subjected to 40 cycles of denaturation (at 95°C for 30 seconds), annealing (at 62°C for 30 seconds), and elongation (at 72°C for 30 seconds) using a Techne TC-412 thermal cycler. In a total volume of 20 μL, 10 μL of the amplicons were digested with 1 unit of the BstU-I restriction endonuclease (New England Biolabs, Hitchin, UK) at 60°C overnight. The digest fragments were 135, 82, and 25 bp for the C allele and 160 and 82 bp for the T allele variant. The fragments were resolved by electrophoresis on a 3.5% agarose gel after staining with ethidium bromide.

Histology.

As mentioned above, 144 out of 211 patients (68.2%) underwent a liver biopsy before starting therapy. Grade and stage were scored according to the Ishak system.17

Antiviral Therapy Schedule and Outcomes.

All patients were treated with a combination therapy of PEG-IFN plus ribavirin. One hundred fifty-three patients (72.5%) received peginterferon alfa-2b (PegIntron, Schering-Plough, New Jersey, USA) at a dosage of 1.5 μg/kg/week, and 58 patients (27.5%) received peginterferon alfa-2a (Pegasys, Roche, Basel, Switzerland) at a dosage of 180 μg per week. In patients infected with HCV genotypes 1, 4, and 5, ribavirin (either Rebetol, Schering-Plough, or Copegus, Roche) was administered according to body weight (1,000 mg/day for patients weighing <75 kg, 1,200 mg/day for patients weighing ≥75 kg); in the case of infection by genotypes 2 and 3, a single ribavirin dose of 800 mg/day was used. The duration of therapy was 48 weeks for genotypes 1, 4, and 5 and 24 weeks for genotypes 2 and 3. Rapid viral response (RVR) was defined as an undetectable serum HCV RNA (<50 IU/mL) level 4 weeks after starting therapy. Complete early viral response (cEVR) was defined as an undetectable serum HCV RNA level 12 weeks after starting therapy. The end of treatment viral response (EOT) was defined as an undetectable serum HCV RNA level after completing the treatment schedule. Sustained viral response (SVR) was defined as an undetectable serum HCV RNA level at 24 weeks after stopping antiviral therapy. Patients who achieved EOT but reverted to a detectable HCV RNA level after stopping therapy were considered relapsers. Dropout was defined as discontinuation of antiviral therapy due to adverse effects. The stopping rule consisted of therapy discontinuation in HCV 1-, 4- and 5-infected patients who either failed to obtain a reduction in serum HCV RNA concentration of at least 2 log compared with baseline at week 12 or had a detectable serum HCV RNA level after 24 weeks of therapy.18-20 Patients who met stopping rule criteria for therapy discontinuation were defined as nonresponders.

Follow-up.

Clinical evaluation of patients, including physical examination and complete blood count (CBC), was performed weekly for the first 4 weeks of therapy and monthly thereafter. Quantitative HCV RNA and alanine aminotransferase levels were evaluated at 4, 12, 48, and 72 weeks after the start of antiviral therapy. An autoimmune panel and thyroid function tests were checked every 3 months.

Response to Antiviral Therapy.

The primary endpoint was the achievement of an SVR. Secondary endpoints were the achievement of cEVR and EOT. According to an intention-to-treat analysis, patients who discontinued antiviral therapy due to adverse events were considered nonresponders.

Statistical Analysis.

Statistical analysis of the data was performed using the BMDP dynamic statistical software package 7.0 (Statistical Solutions, Cork, Ireland). Continuous variables are presented as the median (range) and categorical variables are presented as frequencies (%). The associations between categorical variables were evaluated using a Pearson chi-squared test and, when appropriate, a chi-squared test for linear trend. The chi-squared G test (goodness of fit) was employed to verify whether the proportions of the IL-28B rs12979860 C/T polymorphism genotypes were distributed in accordance with the Hardy-Weinberg equation. Stepwise logistic regression analysis with a forward approach was performed to identify independent predictors of SVR.

Results

Viral Response.

RVR was achieved by 105 patients (49.8%), cEVR was achieved by 153 (72.5%) patients, EOT was achieved by 160 (75.8%) patients, and SVR was achieved by 134 (63.5%) patients. Table 2 shows the detailed rates of responses according to the different HCV genotypes. The association between the achievement of an SVR and the main clinical and demographic variables known to influence HCV viral clearance is reported in Table 3. Univariate analysis showed that the SVR rate was influenced by viral genotype, IL-28B rs12979860 C/T polymorphism, baseline serum cholesterol, and gamma-glutamyl transpeptidase (GGT) levels; it was also influenced by having received a cumulative dose of IFN and ribavirin exceeding 80% of the scheduled dose.

Table 2. Rates of Rapid, Early, Complete Early, End of Treatment, and Sustained Viral Response According to HCV Genotype
 Hepatitis C Virus Genotype
1234-5
  1. Abbreviation: HCV, hepatitis C virus.

Total95 (45.0%)63 (29.9%)38 (18.0%)15 (7.1%)
Rapid viral response26 (27.4%)43 (68.3%)32 (84.2%)4 (26.7%)
Complete early viral response48 (50.5%)59 (93.7%)37 (97.4%)9 (60.0%)
End of treatment viral response57 (60.0%)57 (90.5%)37 (97.4%)9 (60.0%)
Sustained viral response39 (41.1%)54 (85.7%)33 (86.8%)8 (53.3%)
Nonresponders28 (29.5%)0 (0.0%)0 (0.0%)6 (40.0%)
Relapsers18 (18.9%)3 (4.8%)4 (10.5%)1 (6.7%)
Dropouts11 (11.6%)8 (12.7%)1 (2.6%)3 (20.0%)
Table 3. Baseline Sustained Viral Response Demographic and Clinical Predictors in Chronic HCV-Infected Patients Treated with Pegylated Interferon and Ribavirin
 SVR+ (n = 134)SVR– (n = 77)P Value
  • Abbreviations: ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; HCV, hepatitis C virus; HOMA, homeostasis model assessment; IL-28B, interleukin-28B; SVR, sustained viral response.

  • The statistical analysis was performed by means of Pearson chi-squared test or chi-squared test for linear trend when appropriate.

  • *

    Available in 144 patients.

Age >50 years59 (44.0%)33 (42.9%)0.869
Male sex68 (50.7%)41 (53.2%)0.726
Body mass index >25 kg/m249 (36.6%)34 (44.2%)0.277
HOMA index  0.558
 <2.069 (51.5%)37 (48.1%) 
 2.0-4.038 (28.4%)22 (28.6%) 
 >4.027 (20.1%)18 (23.4%) 
Cholesterol ≤200 mg/dL98 (73.1%)69 (89.6%)0.005
ALT >60 IU/mL76 (56.7%)47 (61.0%)0.540
GGT >60 IU/mL31 (23.1%)33 (42.9%)0.003
HCV RNA >600,000 IU/mL74 (55.2%)49 (63.6%)0.233
HCV genotype 1/4/547 (35.1%)63 (81.8%)<0.001
Use of peginterferon alfa-2b91 (67.9%)58 (75.3%)0.255
Grading pretreatment >4*17 (18.3%)12 (23.5%)0.453
Staging pretreatment >2*35 (37.6%)26 (51.0%)0.121
Ribavirin dose per kg body weight ≤13 g46 (34.3%)21 (27.3%)0.289
Cumulative dose of ribavirin ≤80%11 (8.2%)19 (24.7%)0.001
Cumulative dose of interferon ≤80%11 (8.2%)14 (18.2%)0.031
Alcohol consumption >20 g/day16 (11.9%)8 (10.4%)0.733
IL-28B rs12979860 C/T polymorphism  0.004
 C/C60 (44.8%)16 (20.8%) 
 C/T60 (44.8%)51 (66.2%) 
 T/T14 (10.4%)10 (13.0%) 

25-OH Vitamin D Levels.

The median value of circulating vitamin D was 20.7 ng/mL (range, 2.1-59.6). One hundred thirteen (53.6%) patients had normal (>20 ng/mL) 25-OH vitamin D serum levels; 98 (46.4%) patients had a vitamin D deficiency (≤20 ng/mL); and 34 (16.1%) patients had a severe deficiency (≤10 ng/mL). Table 4 displays the possible associations between several clinical and demographic variables and serum vitamin D concentration, categorized according to the above-defined cutoff values. Multivariate analysis, performed using stepwise logistic regression, identified independent predictors of low vitamin D serum levels (≤20 ng/mL) to be age >50 years (odds ratio [OR] 2.37, 95% confidence interval [CI] 1.34-4.21; P = 0.002) and having drawn the blood sample for vitamin D measurement during the winter or spring months (OR 2.06, 95% CI 1.15-3.67; P = 0.016). When the analysis was performed in the subgroup of the 144 patients who underwent a pretreatment liver biopsy, a higher histology grade (OR 3.42, 95% CI 1.37-8.54; P = 0.004) and having drawn the blood sample for vitamin D measurement during the winter or spring months (OR 2.79, 95% CI 1.35-5.74; P = 0.005) were the only independent predictors of low vitamin D serum levels.

Table 4. Relationship Between Clinical and Demographic Variables and Baseline Serum 25-OH Vitamin D Level
 Serum 25-OH Vitamin D LevelP Value
≤10 ng/mL (n = 34)>10/≤20 ng/mL (n = 64)>20 ng/mL (n = 113)
  • Abbreviations: ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; HCV, hepatitis C virus; HOMA, homeostasis model assessment; IL-28B, interleukin-28B.

  • The statistical analysis was performed using a chi-squared test for linear trend.

  • *

    Available in 144 patients.

Age >50 years23 (67.6%)31 (48.4%)38 (33.6%)<0.001
Female sex24 (70.6%)31 (48.4%)47 (41.6%)0.005
Body mass index >25 kg/m213 (38.2%)28 (43.7%)42 (37.2%)0.695
HOMA index >4.07 (20.6%)9 (14.1%)29 (25.7%)0.246
Cholesterol <200 mg/dL28 (82.4%)44 (68.7%)95 (84.1%)0.310
ALT >60 IU/mL23 (67.6%)35 (54.7%)65 (57.5%)0.448
GGT >60 IU/mL11 (32.4%)22 (34.4%)31 (27.4%)0.426
HCV RNA >600,000 IU/mL13 (38.2%)42 (65.6%)68 (60.2%)0.094
HCV genotype 1/4/517 (50.0%)34 (53.1%)59 (52.2%)0.880
Grading pretreatment >4*11 (55.0%)9 (20.0%)9 (11.4%)<0.001
Staging pretreatment >2*16 (76.2%)16 (36.4%)29 (36.7%)0.008
Blood sampling during winter/spring26 (76.5%)37 (57.8%)54 (47.8%)0.003
Alcohol consumption >20 g/day4 (11.8%)8 (12.5%)12 (10.6%)0.775
IL-28B rs12979860 C/T or T/T genotypes26 (76.5%)39 (60.9%)70 (61.9%)0.208

25-OH Vitamin D and Viral Response.

When taking into account all HCV genotypes, a direct relationship was observed between higher 25-OH vitamin D serum levels and a higher rate of treatment response (Table 5). In particular, 25-OH vitamin D serum levels were strongly associated with the rates of RVR and cEVR. In multivariate analysis, 25-OH vitamin D serum levels >20 ng/mL predicted an SVR independent of the other well-known predictors of viral response reported in Table 3. Considering all genotypes, the final model of viral response prediction included HCV genotype, the IL-28B rs12979860 C/T polymorphism, GGT, HCV RNA, cholesterol, and 25-OH vitamin D, with an area under the receiver operating characteristic (ROC) curve of 0.827 (Table 6). When difficult-to-treat genotypes were analyzed separately, even stronger associations were detected between serum vitamin D levels and the rate of viral response (Table 5). Moreover, the serum vitamin D level was confirmed to be an independent predictor of an SVR. In difficult-to-treat genotypes, the final model of viral response prediction included the IL-28B rs12979860 C/T polymorphism, viral load, and serum vitamin D level, with an area under the ROC curve of 0.836 (Table 6).

Table 5. Rate of Rapid, Early, Complete Early, End of Treatment, and Sustained Viral Response in Relationship to Baseline 25-OH Vitamin D Level
 25-OH Vitamin D Level
All HCV GenotypesHCV Genotypes 1/4/5
≤10 ng/mL (n = 34)>10/≤20 ng/mL (n = 64)>20 ng/mL (n = 113)P Value≤10 ng/mL (n = 17)>10/≤20 ng/mL (n = 34)>20 ng/mL (n = 59)P Value
  1. Abbreviation: HCV, hepatitis C virus.

  2. Data are presented in all patients without stratifying for the HCV genotype, and in the subgroup of difficult-to-treat 1-4-5 genotypes. The statistical analysis was performed using a chi-squared test for linear trend.

Rapid viral response10 (29.4%)28 (43.7%)67 (59.3%)0.0011 (5.9%)4 (11.8%)25 (42.4%)<0.001
Complete early viral response19 (55.9%)43 (67.2%)91 (80.5%)0.0024 (23.5%)14 (41.2%)39 (66.1%)<0.001
End of treatment viral response18 (52.9%)49 (76.6%)93 (82.3%)0.0015 (29.4%)20 (58.8%)41 (69.5%)0.004
Sustained viral response17 (50.0%)39 (60.9%)78 (69.0%)0.0384 (23.5%)11 (32.4%)32 (54.2%)0.009
Nonresponders10 (29.4%)10 (15.6%)14 (12.4%)0.02810 (58.8%)10 (29.4%)14 (23.7%)0.012
Relapsers1 (2.9%)10 (15.6%)15 (13.3%)0.2311 (5.9%)9 (26.5%)9 (15.3%)0.799
Dropouts5 (14.7%)8 (12.5%)10 (8.8%)0.2851 (5.9%)7 (20.6%)6 (10.2%)0.894
Table 6. Stepwise Logistic Regression Analysis in Predicting Sustained Viral Response in All HCV Genotypes and in HCV Genotypes 1, 4, and 5
 All HCV GenotypesHCV Genotypes 1/4/5
OR95% CIP ValueOR95% CIP Value
  1. Abbreviations: CI, confidence interval; GGT, gamma-glutamyl transpeptidase; HCV, hepatitis C virus; IL-28B, interleukin-28B; OR, odds ratio.

  2. The variables included were those reported in Table 3 (except for histology available in 144 patients) and 25-OH vitamin D serum levels.

HCV genotypes 1/4/50.1190.057-0.248<0.001
IL-28B rs12979860 C/T polymorphism  0.008  <0.001
 C/T0.3310.151-0.724 0.1100.034-0.352 
 T/T0.5890.187-1.860 0.1990.043-0.922 
GGT >60 IU/mL0.4760.230-0.9840.033
HCV RNA >600,000 IU/mL0.4450.216-0.9140.0290.2020.070-0.5800.003
Cholesterol >200 mg/dL3.1801.180-8.5700.030
25-OH vitamin D >20 ng/mL2.0701.020-4.1700.0402.8101.060-7.4900.040

IL-28B rs12979860 C/T Polymorphism and Viral Response.

The following allelic and genotypic frequencies for the IL-28B rs12979860 C/T polymorphism were detected: C, 0.623; T, 0.377; C/C, 76 (36.0%); C/T, 111 (52.6%); and T/T, 24 (11.4%). Genotypic frequencies did not differ from what was expected based on the Hardy-Weinberg equilibrium equation (P > 0.05). Considering all of the patients, the SVR rate occurred as follows: C/C, 60/76 (78.9%); C/T, 60/111 (54.1%); and T/T, 14/24 (58.3%) (P = 0.004). In difficult-to-treat HCV genotypes, the SVR rates were: C/C, 24/32 (75.0%); C/T, 16/61 (26.2%); and T/T, 7/17 (41.2%) (P = 0.002).

Combined Assessment of Serum Vitamin D Levels and the IL-28B rs12979860 C/T Polymorphism.

To verify the usefulness of the combined assessment of the IL-28B rs12979860 C/T polymorphism and the serum vitamin D level, we identified four groups of patients: C/C homozygotes with vitamin D levels of >20 ng/mL (group A), C/C homozygotes with vitamin D levels of ≤20 ng/mL (group B), C/T heterozygotes and T/T homozygotes with vitamin D levels of >20 ng/mL (group C), and C/T heterozygotes and T/T homozygotes with vitamin D levels of ≤20 ng/mL (group D). Considering the whole cohort of treated patients, a highly significant linear trend was found in the decrease of the SVR rate: group A, 35/43 (81.4%); group B, 25/33 (75.8%); group C, 43/70 (61.4%); and group D, 31/65 (47.7%) (P = 0.0001). Taking into account only difficult-to-treat HCV genotypes (1, 4, and 5), an even more pronounced stratification of the data occurred: group A, 18/21 (85.7%); group B, 6/11 (54.5%); group C, 14/38 (36.8%); and group D, 9/40 (22.5%) (P < 0.0001). In these patients, the comparisons between groups (group B versus group C, P = 0.293; group C versus group D, P = 0.165) were not significant, although the comparison of group A versus group B was close to statistical significance (P = 0.053).

Accordingly, among the components of the chi-squared test (final value, 23.7), when comparing the SVR rates among the four groups, the major contribution was provided by group A (chi-squared, 15.9; P < 0.01). In patients with easy-to-treat HCV genotypes, no association was detected between the combined assessment of the IL-28B rs12979860 C/T polymorphism and the serum vitamin D level and the rate of SVR achievement: group A, 17/22 (77.3%); group B, 19/22 (86.4%); group C, 29/32 (90.6%); and group D, 22/25 (88.0%) (P = 0.251) (Fig. 1). Because SVR rates were significantly influenced by the interaction between IL-28B genotypes and vitamin D serum levels only in difficult-to-treat HCV genotypes (Fig. 1), further analysis was performed only in this subgroup of patients. Stepwise logistic regression analysis was performed to verify whether the combined assessment of the IL-28B genotype and the serum vitamin D level could be an independent predictor of SVR achievement. The variables included were those listed in Table 3 (except for liver histology due to insufficient data) and pretreatment serum vitamin D level, either alone or in combination with the IL-28B genotype, as in groups A-D. The only independent predictors of SVR selected by the analysis were the combined assessment of the IL-28B genotype and the serum vitamin D level and baseline HCV RNA level, with an area under the ROC curve of 0.854 (Table 7). In the analysis of both pretreatment and in-treatment variables, RVR was the strongest predictor of SVR (OR 22.5, 95% CI 5.16-98.5; P < 0.001).

Figure 1.

Rates of sustained viral response (SVR) in relationship to IL-28B rs12979860 C/T polymorphism and 25-OH vitamin D serum levels. Results are presented in all HCV genotypes and separately in genotypes 1/4/5 and 2/3. Patients are divided into four groups: those carrying the C/C genotype with vitamin D levels >20 ng/mL, those carrying the C/C genotype with vitamin D levels ≤20 ng/mL, those carrying the C/T or T/T genotypes with vitamin D levels >20 ng/mL, and those carrying the C/T or T/T genotypes with vitamin D levels ≤20 ng/mL. Statistical analysis was performed using a chi-squared test for linear trend.

Table 7. Stepwise Logistic Regression Analysis in Predicting Sustained Viral Response in Difficult-to-Treat HCV Genotypes (1, 4, and 5)
 HCV Genotypes 1/4/5
OR95% CIP Value
  1. Abbreviations: CI, confidence interval; HCV, hepatitis C virus; IL-28B, interleukin-28B; OR, odds ratio.

  2. The variables included were those reported in Table 3 (except for histology), 25-OH vitamin D serum levels and its combined assessment with IL-28B rs12979860 C/T polymorphism. Group A: C/C homozygotes with vitamin D levels >20 ng/mL. Group B: C/C homozygotes with vitamin D levels ≤20 ng/mL. Group C: C/T heterozygotes and T/T homozygotes with vitamin D levels >20 ng/mL. Group D: C/T heterozygotes and T/T homozygotes with vitamin D levels ≤20 ng/mL.

IL-28B and vitamin D  <0.001
 Group A1.000 
 Group B0.1050.014-0.792 
 Group C0.0550.010-0.316 
 Group D0.0280.005-0.162 
HCV RNA >600,000 IU/mL0.2090.069-0.6330.003
Cholesterol >200 mg/dL3.580.837-15.30.057

Discussion

Recently, it has been recognized that vitamin D deficiency is common among patients with chronic liver disease. This trend occurs not only in patients with chronic cholestatic liver disease or advanced fibrosis/cirrhosis, where it can be expected, but also in mild chronic hepatitis C.21 In chronic cholestasis, decreased intestinal absorption of vitamin D is a plausible mechanism for vitamin D deficiency, whereas in end stage liver disease, an impaired liver synthesis of 25-OH vitamin D may occur.22 The reasons why vitamin D deficiency occurs in patients with chronic hepatitis C are far less clear. An explanation of this finding likely requires taking into account the multiple interconnections between vitamin D, the immune response, and inflammatory status.23, 24

In agreement with the above reports, in the present study that included only patients with chronic hepatitis C, the occurrence of vitamin D deficiency (≤20 ng/mL) was observed in approximately one-half of the patients and severe vitamin D deficiency (≤10 ng/mL) in approximately 16% of them. On multivariate analysis, only age, time of the year of blood sampling, and grading score were found to be independent predictors of low serum vitamin D levels. Interestingly, the histology staging score, which had a strong inverse association with serum vitamin D level on univariate analysis, did not appear to be an independent predictor of vitamin D deficiency, probably because liver fibrosis parallels age, a strong universally recognized predictor of vitamin D body storage. The results presented in this study conflict with those previously published by Petta et al.,12 who reported an inverse association not only between histology grading and vitamin D but also between the staging of liver fibrosis and the vitamin D level. Nevertheless, using the data from Petta et al., when vitamin D was analyzed as a categorical variable using a cutoff level of 30 ng/mL, the histology grading score further confirmed its predictive role, whereas this result did not occur for the staging score, a finding similar to ours. The bulk of the studies concerning the relevance of vitamin D in the clinical setting have clearly shown that true deficiency, whether mild (≤20 ng/mL) or severe (≤10 ng/mL), is important, whereas simple variations of vitamin D serum levels above the limit of normality seem to have a negligible biological effect. Thus, the choice to analyze vitamin D as a categorical variable appears to be appropriate.

In the present retrospective study, the achievement of SVR ranged from 40% to 50% in patients infected with difficult-to-treat HCV genotypes, whereas the corresponding rate for patients with easy-to-treat genotypes was approximately 85%. Therefore, the SVR rates reported in the present study are comparable to those obtained from the largest clinical trials.19, 20, 25, 26 Moreover, in the present paper, the IL-28B rs12979860 C/T polymorphism was confirmed to play a pivotal role in predicting the rate of SVR independent of the accepted predictors of SVR achievement, such as HCV genotype, HCV viral load, baseline serum cholesterol, and GGT. Thus, two comments may be made. First, the frequencies of IL-28B alleles and genotypes were found to be very similar to those reported for European populations in the landmark paper of Ge et al.8 Second, the rates of SVR found to stratify patients according to the IL-28B rs12979860 C/T polymorphism were not different from those reported in the same paper.

In this study, vitamin D levels were found to influence the achievement of viral clearance after antiviral therapy in patients with chronic HCV infection. In particular, there was a highly significant association between progressively lower baseline serum vitamin D levels and the rates of viral clearance. This outcome was evident in all HCV genotypes but was particularly important in patients infected by difficult-to-treat genotypes, as reported by Petta et al.12 Interestingly, baseline vitamin D affects not only SVR but also the earliest treatment milestones of RVR and cEVR. RVR has been claimed as a strong predictor of SVR in difficult-to-treat HCV genotypes;27, 28 consequently, one might hypothesize that the predictive role of baseline serum vitamin D on the attainment of SVR could derive from the strong association found between vitamin D and RVR attainment. Hypothetically, vitamin D levels might be assessed before starting antiviral therapy, which should be initiated only in the presence of normal serum vitamin D values; in the presence of vitamin D deficiency, it might be preferable to correct the deficiency before starting antiviral therapy. The supposed relationship between the rapid slope of the HCV RNA level after therapy initiation and vitamin D suggests that the latter could amplify the immunological effect of IFN. In fact, beyond the classical actions related to calcium homeostasis and bone metabolism, vitamin D has emerged as a key regulator of the innate immunity response in humans.23, 29, 30

Can pretreatment serum vitamin D determination be a useful adjunct to IL-28B rs12979860 C/T polymorphism evaluation in managing the treatment options for patients with chronic hepatitis C? This study demonstrated that the vitamin D level and the IL-28B rs12979860 C/T polymorphism are two independent predictors of SVR achievement in difficult-to-treat HCV genotypes. Moreover, our results clearly illustrate that these two predictors, being completely independent of each other, may be usefully combined to enhance the ability to identify patients who will respond to treatment. Compared with patients carrying the IL-28B rs12979860 C/C genotype and who have a normal vitamin D level, vitamin D deficiency identifies patients with a lower probability of SVR attainment. Furthermore, carrying at least one T allele along with vitamin D deficiency was associated with the lowest probability of attaining the same viral endpoint.

Although promising, this study has some limitations. First, it is retrospective. Second, only a baseline vitamin D determination was available, and no further vitamin D levels could be included in the analysis. Therefore, we cannot exclude that during antiviral treatment, vitamin D levels vary in relationship with a number of factors capable of influencing its level. However, in accordance with the data presented, vitamin D plays its major role during the initial phases of viral decline soon after initiating treatment, and although dependent on several environmental factors, vitamin D levels are probably at least in part genetically predetermined.31

In conclusion, the present study confirms a possible role for the serum vitamin D level in predicting the outcome of antiviral therapy in HCV chronic infection. Vitamin D deficiency is associated with a reduced probability of RVR attainment. The determination of this vitamin may be complementary to that of the IL-28B rs12979860 C/T polymorphism in enhancing the correct prediction of SVR achievement in treatment-naïve patients with chronic hepatitis C.

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