Patient Response and Genetics: Interferon Lambda 4 Thomas R. O'Brien, MD, MPH Senior Investigator Infections and Immunoepidemiology Branch Division of Cancer Epidemiology and Genetics Disclosure • I am a co-inventor on patent applications filed by the National Cancer Institute for the IFNL4- ΔG genotype and the IFN-λ4 protein Overview • Discovery of IFN-λ4 and IFNL4-ΔG • Functional Studies • IFNL4-ΔG Genotype in the DAA Era Background Haplotype - DNA variants, lying in a chromosomal region, that tend to be inherited together Linkage Disequilibrium • Non-random association of alleles at two or more loci • Correlation (r2) Haplotype Blocks Genome Wide Association Studies (GWAS) • Scan SNPs across complete genome to find associations with a disease or condition • Knowledge of haplotype patterns reduces number of SNPs 10,000,000 → 500,000-1,000,000 • Identified SNPs are usually markers for unknown ‘functional’ genetic variant Strategy for Genomic Discovery Genome Wide Association Study Condition Marker SNP Functional Variant Linkage Disequilibrium (correlation) GWAS Markers for HCV Infection • Highly associated with treatment response – Strongest host factor – Basis for clinical test to predict treatment response • Located in interferon-λ region Ge, Nature 2009 Suppiah, Nature Genetics 2009 Tanaka, Nature Genetics 2009 Rauch, Gastroenterology 2010 Interferon-λ • Cytokine family discovered via computational prediction from genomic sequence • Current gene symbols: – IFNL1 (formerly IL29) – IFNL2 (formerly IL28A) – IFNL3 (formerly IL28B ) Kotenko et al. Nat. Immunol 2003 Sheppard et al. Nat. Immunol 2003 GWAS Marker Near IFNL3 (IL28B) q13.12 q13.13 Chromosome 19 IFNL3 (IL28B) IFNL2 (IL28A) IFNL1 (IL29) GWAS Marker Strategy for Genomic Discovery HCV Clearance HCV Clearance Marker SNP Functional Variant rs12979860 ? Linkage Disequilibrium (correlation) Strategy for Genomic Discovery RNA Sequencing (RNA-seq) • Whole-genome RNA sequencing analyzes both the known and unknown transcriptional landscape • We sequenced mRNA from primary human hepatocytes from a donor who was heterozygous for rs12979860 • Treated with Poly I:C (synthetic mimic of double- stranded RNA) and examined for transcription at 6 time- points Prokunina-Olsson et al. Nature Genetics, 2013 RNA-Seq - IFNL3 (IL28B) Region IFNL3 (IL28B) IFNL4 Prokunina-Olsson et al. Nature Genetics, 2013 Interferon Lambda 4 (IFNL4) rs12979860 Exons: 5 4 3 2 1 rs12979860 variant, which is commonly referred to as ‘IL28B’, is actually located within IFNL4 Prokunina-Olsson et al. Nature Genetics, 2013 Interferon Lambda 4 (IFNL4) ss469415590 (IFNL4-ΔG/TT) Exons: 5 4 3 2 1 Insertion allele (TT) causes frame-shift Only IFNL4-ΔG carriers generate IFN-λ4 protein Prokunina-Olsson et al. Nature Genetics, 2013 IFNL4 Allele Frequencies HapMap Reference Populations Variant, allele Asians Europeans West- Africans IFNL4-ΔG 6.7% 32.4% 76.7% Prokunina-Olsson et al. Nature Genetics, 2013 Linkage Disequilibrium (r2) HapMap Reference Populations Variant, allele Asians Europeans West- Africans IFNL4-ΔG - - - rs12979860-T 1.00 0.92 0.71 Prokunina-Olsson et al. Nature Genetics, 2013 Virahep-C • NIDDK sponsored clinical trial of ~400 patients • Compared treatment response to pegylated IFN-α/ribavirin in African-American patients with chronic hepatitis C to otherwise similar patients of European ancestry HCV RNA Decline in Response to Peg-IFN-α/Ribavirin Virahep-C, African Americans P=0.015 p=0.015 Nature Genetics, 2013 TT/-G is a better predictor of response to treatment than rs12979860. Bibert S et al. J Exp Med 2013;210:1109-1116 © 2013 Bibert et al. Clinical Implication • For patients of African or European ancestry, IFNL4- ΔG genotype is superior to rs12979860 (current clinical test) for predicting response to treatment, while providing identical information in patients of Asian ancestry Strategy for Genomic Discovery HCV Clearance HCV Clearance Marker SNP Functional Variant rs12979860 IFNL4-ΔG Linkage Disequilibrium (correlation) Overview • Discovery of IFN-λ4 and IFNL4-ΔG • Functional Studies • IFNL4-ΔG Genotype in the DAA Era O’Brien, Nature Genetics, 2009 Comparison of IFN-λ4 to Type-2 Cytokines Protein IFN-λ4 IFN-λ3 IFN-λ1 IFN-α1 Size, aa 179 196 200 189 Exons 5 5 5 1 Identity, - 29% 27% 15% aa (%) IFN-λ4 dissimilar to IFN-λ3 in IL10R2 binding region Prokunina-Olsson et al. Nature Genetics, 2013 IFN-λ4 Function IFNL4-ΔG (‘IL28B’-T) Associations • Prior to treatment – Lower baseline HCV RNA levels – Greater expression of interferon stimulated genes (ISGs) - suggests activation of JAK-STAT pathway • Response to IFN-α treatment – Less increase in ISG expression – Less decrease in HCV RNA – Lower rate of SVR Ge, Nature, 2009 Honda, Gastroenterology, 2010 Urban, Hepatology, 2010 STAT Phosphorylation IFN-λ4 Transfected HepG2 Cells IFNL3 IFNL4 Prokunina-Olsson et al. Nature Genetics, 2013 IFN-λ4 Induces Expression of ISGs Transfection with an IFN-λ4 expression construct induced activation of an interferon stimulated response element reporter (ISRE-Luc) Prokunina-Olsson et al. Nature Genetics, 2013 IFN-λ4 Decreases HCV Replication Transient transfection with IFN-λ4 construct → ↓HCV RNA replication in hepatoma cells expressing luciferase-expressing HCV replicon Prokunina-Olsson et al. Nature Genetics, 2013 IFN-λ4 Functional Mechanism • Receptor complex unknown • IFN-λ4 activates JAK-STAT pathway • IFN-λ4 impairs response to IFN-α Hypothesis: IFN-λ4 induced signaling → refractory IFN-α signaling Overview • Discovery of IFN-λ4 and IFNL4-ΔG • Functional Studies • IFNL4-ΔG Genotype in the DAA Era IFNL4 Genotype and Response to Peg-INF/Ribavirin plus Telaprevir Akuta Hepatology, 2010 HCV RNA Decline in Response to IFN-Free Regimen, by rs12979860 Genotype HCV Genotype 1 p=0.009 Chu, Gastroenterology 2012 HCV RNA Decline in Response to Sofosbuvir/RBV, by IFNL4-ΔG Genotype HCV Genotype 1 p=0.03 ΔG/ΔG (n=8) TT/ΔG (n=8) TT/TT (n=4) Meissner, Int’l Liver Congress, 2013 SVR with Sofosbuvir/RBV, by rs12979860 Genotype HCV Genotype 2 or 3 Jacobson et al. NEJM 2013 SVR with Sofosbuvir/PEG-INFα/RBV or Sofosbuvir/RBV, by rs12979860 Genotype HCV Genotype 1 or 4 HCV Genotype 2 or 3 Lawitz et al. NEJM 2013 Summary 1) The rs12979860 variant, commonly referred to as ‘IL28B’, is actually located within IFNL4 2) rs12979860-T is strongly correlated with IFNL4-ΔG 3) Only IFNL4-ΔG carriers generate the IFN-λ4 protein 4) In African American and European patients, IFNL4- ΔG genotype predicts peg-IFN-α/ribavirin treatment response better than the ‘IL28B’ genotype Summary 5) IFNL4-ΔG appears to be the functional variant underlying GWAS findings, however, the functional mechanism is not yet known 6) The clinical role of IFNL4-ΔG genotype in the DAA era remains to be determined Collaborators NCI NIDDK IIB Barbara Rehermann Dianna Buckett Heiyoung Park Alan Wang FDA – Div Therapeutic Proteins Biostatistics Branch Raymond P. Donnelly Ruth Pfeiffer Lab. of Translational Genomics Virahep-C Ludmila Prokunina-Olsson Charles D. Howell Brian Muchmore Wei Tang HALT-C NIAID Herbert L. Bonkovsky Shyam Kottilil Timothy R. Morgan Eric Meissner .