Volume 140, Issue 3, Pages 1021-1031.e10 (March 2011) Interferon-Induced Gene Expression Is a Stronger Predictor of Treatment Response Than IL28B Genotype in Patients With Hepatitis C  Michael T. Dill, Francois H.T. Duong, Julia E. Vogt, Stéphanie Bibert, Pierre–Yves Bochud, Luigi Terracciano, Andreas Papassotiropoulos, Volker Roth, Markus H. Heim  Gastroenterology  Volume 140, Issue 3, Pages 1021-1031.e10 (March 2011) DOI: 10.1053/j.gastro.2010.11.039 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Association of IL28B SNPs with SVR and hepatic IL28B mRNA expression. (A) Percentage of SVR in different IL28B genotypes for the SNPs rs12979860 and rs8099917. The minor allele frequencies (MAF) for each SNP are indicated. Association of the major allele with SVR was assessed by a logistic regression model and is statistically significant for each SNP. (B) The minor IL28B allele correlates with lower expression of IL28B mRNA in the liver of patients with CHC. IL28B mRNA was determined by quantitative reverse-transcription polymerase chain reaction and normalized relative to GAPDH mRNA. Shown are the mean values (±SEM) after log transformation. The P values were obtained with Student t test. The IL28B genotypes for the rs12979860 and the rs8099917 SNPs and the number of patients in each group are shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 The expression of 8 classifier genes differs significantly between SVR, nonresponse, and relapse. Aligned dot plot of hepatic gene expression in patients with CHC stratified according to response to treatment (SVR, n = 33; relapse [REL], n = 9; nonresponse [NR], n = 31). Relative hepatic mRNA was assessed with quantitative polymerase chain reaction for (A) the 8 classifier genes (IFI44L, RSAD2, ISG15, IFI27, LAMP3, OAS3, LGALS3BP, and HTATIP2) and (B) genes from the IL28 cytokine family (IL28A, IL28B, IL29) and the IL28 receptor chains (IL28RA, IL10RB). Values are shown after log transformation and normalization to GAPDH. Each dot represents one sample. Mean ± SEM are shown. P values were obtained with Student t test. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Hepatic ISG expression according to IL28B genotypes (SNP = rs12979860). (A) Expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in the liver according to the IL28B genotype (rs12979860) in 109 patients with CHC. (B) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 19 patients with the rs12979860 CC genotype, stratified according to treatment response (SVR vs non-SVR). (C) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 47 patients with the rs12979860 CT genotype, stratified according to treatment response (SVR vs non-SVR). Shown are mean values (±SEM) after log transformation. P values were obtained with Student t test. The number of patients in each group is shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Hepatic ISG expression according to IL28B genotypes (SNP = rs12979860) in patients infected with HCV genotype 1 or 4. (A) Expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in the liver according to the IL28B genotype (rs12979860) in 75 patients with CHC infected with HCV genotype 1 or 4. (B) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 10 patients with rs12979860 CC genotype, stratified according to treatment response (SVR vs non-SVR). (C) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 32 patients with rs12979860 CT genotype, stratified according to treatment response (SVR vs non-SVR). Shown are mean values (±SEM) after log transformation. P values were obtained with Student t test. The number of patients in each group is shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Hepatic expression of IFI27, ISG15, RSAD2, and HTATIP2 is a better predictor of treatment response than IL28B allelic variants. (A) Importance plot of all variables in the entire 73 patient dataset (including all HCV genotypes). (B) Importance plot of all variables in the 45 treated patients with HCV genotypes 1 and 4. The RFFS is a measure of the predictive power of the variables for separating the classes. The importance of the variables is computed by permuting the values of a variable m in the out-of-bag case and putting these cases down the decision tree. The number of votes for the correct class in the permuted out-of-bag set is subtracted from the number of votes for the correct class in the untouched out-of-bag-set and averaged over all trees in the forest. This gives the importance score for variable m. Note that the importance score is computed on the out-of-bag set; hence, overfitting can occur. Negative importance scores imply that such overfitting phenomena indeed occur for some variables. (C) ROC curve for prediction of treatment response using the rs12979860 SNP (AUC, 0.76; ERR, 0.16) or the rs8099917 SNP (AUC, 0.58; ERR, 0.31) in patients with HCV genotype 1 or 4. (D) ROC curve with the combination of the rs12979860 and rs8099917 SNPs in patients with HCV genotypes 1 and 4 (AUC, 0.73; ERR, 0.16). (E) ROC curve for prediction of treatment response with a combination of IFI27, ISG15, RSAD2, and HTATIP2 in the entire 73 patient dataset (AUC, 0.90; ERR, 0.12) or in the patients infected with HCV genotype 1 or 4 (AUC, 0.92; ERR, 0.09). (F) ROC curve using the combination of IFI27, ISG15, RSAD2, HTATIP2, and the rs12979860 SNP in patients with HCV genotypes 1 and 4 (AUC, 0.90; ERR, 0.13). Adding the IL28B genotype information to the 4-gene classifier impairs the performance of the classifier because of an increase in the complexity of the model without additional information gain (overfitting phenomenon). Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Prediction of RVR in patients infected with HCV genotype 1 or 4. (A) Importance blot of all variables in the 50 patients infected with HCV genotype 1 or 4 and with RVR data available. (B) ROC curve with the rs12979860 SNP for prediction of RVR (AUC, 0.60; ERR, 0.12). (C) ROC curve using a combination of IFI27, ISG15, RSAD2, HTATIP2, and LAMP3 as a 5-gene classifier to predict RVR (AUC, 0.94; ERR, 0.04). (D) ROC curve with the combination of IFI27, ISG15, RSAD2, HTATIP2, LAMP3, and the rs12979860 SNP to predict RVR (AUC, 0.94; ERR, 0.06). Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Association of IL28B SNPs with hepatic mRNA expression of (A) IL28A, (B) IL29 (IFNλ1), (C) IL28RA, and (D) IL10Rβ. Expression levels were determined by quantitative reverse-transcription polymerase chain reaction and normalized relative to GAPDH mRNA. Shown are the mean values (±SEM) after log10 transformation. The only statistical significant differences (Student t test) were found for IL28RA between the rs12979860 CC and CT groups and the rs8099917 TT and TG groups. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Hepatic ISG expression according to IL28B genotypes (SNP = rs8099917). (A) Expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in the liver according to the IL28B genotype (rs8099917) in 109 patients with CHC. (B) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 35 patients with rs8099917 TT genotype, stratified according to treatment response (SVR vs non-SVR). (C) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 38 patients with rs8099917 TG genotype, stratified according to treatment response (SVR vs non-SVR). Shown are mean values (±SEM) after log transformation. P values were obtained with Student t test. The number of patients in each group is shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Hepatic ISG expression according to IL28B genotypes (SNP = rs8099917) in patients infected with HCV genotype 1 or 4. (A) Expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in the liver according to the IL28B genotype (rs8099917) in 75 patients with CHC infected with HCV genotype 1 or 4. (B) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 19 patients with rs8099917 TT genotype, stratified according to treatment response (SVR vs non-SVR). (C) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 26 patients with rs8099917 TG genotype, stratified according to treatment response (SVR vs non-SVR). Shown are mean values (±SEM) after log transformation. P values were obtained with Student t test. The number of patients in each group is shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 (A) Expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in the liver according to the IL28B genotype (rs12979860) in the subset of 73 patients with CHC who have undergone treatment with pegIFN-α and ribavirin. (B) Hepatic expression of 4 ISGs (IFI44L, RSAD2, ISG15, IFI27) in 7 patients with the rs12979860 TT genotype, stratified according to treatment response (SVR vs non-SVR). Due to the small number of patients, the difference is not significant. Shown are mean values (±SEM) after log transformation. P values were obtained with Student t test. The number of patients in each group is shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 5 (A) Expression of 2 ISGs (LAMP3, OAS3) in the liver according to the IL28B genotype (rs12979860) in all 109 patients with CHC and in patients with either CC, CT, or TT IL28B genotype stratified according to treatment response (SVR vs non-SVR). (B) Expression of 2 ISGs (LAMP3, OAS3) in the liver according to the IL28B genotype (rs12979860) in all 75 patients infected with HCV genotype 1 or 4 and in patients with either CC or CT IL28B genotype stratified according to treatment response (SVR vs non-SVR). Shown are mean values (±SEM) after log transformation. P values were obtained with Student t test. The number of patients in each group is shown below the plots. Gastroenterology 2011 140, 1021-1031.e10DOI: (10.1053/j.gastro.2010.11.039) Copyright © 2011 AGA Institute Terms and Conditions