Paul J. Clark

IL28B genomic-based treatment paradigms for patients with chronic hepatitis C infection: The future of personalized HCV therapies

Genome-wide association studies (GWAS) have recently identified host genetic variation to be critical for predicting treatment response and spontaneous clearance in patients infected with hepatitis C virus (HCV). These important new studies are reviewed and their future clinical implications discussed. Single-nucleotide polymorphisms in the region of the IL28B gene on chromosome 19, coding for the interferon (IFN)-λ-3 or IL28B gene, are strongly associated with treatment response to pegylated IFN and ribavirin in patients infected with genotype 1 HCV. The good response variant is associated with a twofold increase in the rate of cure. Allele frequencies differ between ethnic groups, largely explaining the observed differences in response rates between Caucasians, African Americans and Asians. IL28B polymorphism is also strongly associated with spontaneous clearance of HCV. The biological mechanisms responsible for these genetic associations remain unknown and are the focus of ongoing research. Knowledge of a patients IL28B genotype is likely to aid in clinical decision making with standard of care regimens. Future studies will investigate the possibility of individualizing treatment duration and novel regimens according to IL28B type.

Beneficial IL28B genotype associated with lower frequency of hepatic steatosis in patients with chronic hepatitis C.

BACKGROUND & AIMS IL28B polymorphisms have been associated with both treatment induced and spontaneous clearance of hepatitis C virus (HCV). We previously found that LDL cholesterol levels were higher in chronic hepatitis C (CHC) patients with the CC genotype at the rs12979860 polymorphism, located proximal to the IL28 gene. Here we analyzed the association of steatosis with IL28B genotype in treatment naïve patients with CHC. METHODS Two independent cohorts of 145 genotype 1 infected patients from an antifibrotic study and 180 genotype 1 patients from Duke were analyzed for the presence and severity of steatosis in relation to the rs12979860 polymorphism at the IL28B locus. TaqMan assay based genotyping classified three groups CC, CT, and TT. RESULTS CC genotype was associated with a lower prevalence of steatosis. In the antifibrotic study, steatosis was found in 47.6% (50/105) of IL28B non-CC vs. 22.5% (9/40; p=0.008) in CC patients. Similarly, steatosis was found in 67.4% (89/132) of non-CC patients compared to only 39.6% (19/48; p=0.001) of CC patients in the Duke cohort. CONCLUSIONS IL28B CC genotype is associated with less pronounced disturbances of lipid metabolism, as reflected both in serum lipoprotein levels and hepatic steatosis, in HCV infection.

Inosine Triphosphatase Genetic Variants are Protective Against Anemia During Antiviral Therapy for HCV2/3 But Do Not Decrease Dose Reductions of RBV Or Increase SVR

Two functional variants in the inosine triphosphatase (ITPA) gene causing inosine triphosphatase (ITPase) deficiency protect against ribavirin (RBV)‐induced hemolytic anemia and the need for RBV dose reduction in patients with genotype 1 hepatitis C virus (HCV). No data are available for genotype 2/3 HCV. We evaluated the association between the casual ITPA variants and on‐treatment anemia in a well‐characterized cohort of genotype 2/3 patients treated with variable‐duration pegylated interferon alfa‐2b (PEG‐IFN‐α2b) and RBV. Two hundred thirty‐eight Caucasian patients were included in this retrospective study [185 (78%) with genotype 2 and 53 (22%) with genotype 3]. Patients were treated with PEG‐IFN‐α2b plus weight‐based RBV (1000/1200 mg) for 12 (n = 109) or 24 weeks (n = 129). The ITPA polymorphisms rs1127354 and rs7270101 were genotyped, and an ITPase deficiency variable was defined that combined both ITPA variants according to their effect on ITPase activity. The primary endpoint was hemoglobin (Hb) reduction in week 4. We also considered Hb reduction over the course of therapy, the need for RBV dose modification, and the rate of sustained virological response (SVR). The ITPA variants were strongly and independently associated with protection from week 4 anemia (P = 10−6 for rs1127354 and P = 10−7 for rs7270101). Combining the variants into the ITPase deficiency variable increased the strength of association (P = 10−11). ITPase deficiency protected against anemia throughout treatment. ITPase deficiency was associated with a delayed time to an Hb level < 10 g/dL (hazard ratio = 0.25, 95% confidence interval = 0.08‐0.84, P = 0.025) but not with the rate of RBV dose modification (required per protocol at Hb < 9.5 g/dL). There was no association between the ITPA variants and SVR. Conclusion: Two ITPA variants were strongly associated with protection against treatment‐related anemia in patients with genotype 2/3 HCV, but they did not decrease the need for RBV dose reduction or increase the rate of SVR. (HEPATOLOGY 2011;53:389‐395.)

Dysregulation of innate immunity in hepatitis C virus genotype 1 IL28B‐unfavorable genotype patients: Impaired viral kinetics and therapeutic response

Recent studies have shown that a single‐nucleotide polymorphism upstream of the interleukin‐28B (IL28B) gene plays a major role in predicting therapeutic response in hepatitis C virus (HCV)‐infected patients treated with pegylated interferon (PEG‐IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics, IFN pharmacokinetics, IFN pharmacodynamics, and gene expression profiles. Two prospective cohorts (human immunodeficiency virus [HIV]/HCV‐coinfected and HCV‐monoinfected) completing treatment with IFN/ribavirin were enrolled. Patients were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort, frequent serum sampling was completed for HCV RNA and IFN levels. DNA microarray of peripheral blood mononuclear cells and individual expression of IFN‐stimulated genes (ISGs) were quantified on IFN therapy. The IL28B‐favorable (CC) genotype was associated with improved therapeutic response compared with unfavorable (CT or TT) genotypes. Patients with a favorable genotype had greater first‐ and second‐phase viral kinetics (P = 0.004 and P = 0.036, respectively), IFN maximum antiviral efficiency (P = 0.007) and infected cell death loss (P = 0.009) compared with unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of natural killer cells, from patients with unfavorable genotypes (P <0.004). Induction of innate immunity genes was also lower in unfavorable genotypes. ISG expression at baseline and induction with IFN was independent of IL28B genotype. Conclusion: Carriers of the IL28B‐favorable genotype were more likely to have superior innate immune response to IFN therapy compared with unfavorable genotypes, suggesting that the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of ISG expression. (HEPATOLOGY 2012)

Natural killer inhibitory receptor expression associated with treatment failure and interleukin‐28B genotype in patients with chronic hepatitis C

Natural killer (NK) cells constitute a first line of defense against viral infections; their function is governed by the integration of signals from multiple activating and inhibitory surface receptors. We hypothesized that because NKs become rapidly activated by cytokines, response to anti‐hepatitis C virus (HCV) therapy would be predicted by the phenotype and function of NKs. We used a cohort of 101 patients (55 African, 46 Caucasian‐American) who received pegylated‐interferon (IFN) and ribavirin for 48 weeks. Multiparameter FACS analysis was used to examine relative expression of 14 different inhibitory/activating receptors. Interleukin (IL)‐28B genotyping (rs12979860) was also performed. Pretreatment levels of inhibitory receptors CD158a, CD158b, and CD158e were higher in patients who demonstrated poor viral decline within the first 28 days of therapy. Higher expression levels of inhibitory receptors NKG2A, CD158b, and CD158e were demonstrable in patients who failed to achieve sustained virologic response (SVR). Patients carrying the IL‐28B T allele had higher NKG2A expression on effector NKs. We created a mathematical regression model incorporating race, viral level, and two inhibitory receptors. The area‐under‐the curve was 0.88, which is highly predictive of SVR. Moreover, the model performed complementarily with IL‐28B across the CC, CT, and TT genotypes. Purified NKG2Aneg NKs treated with pegylated‐IFN‐α for 4 hours demonstrated higher levels of IFN‐γ‐inducible protein‐10 (IP‐10) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) compared with their NKG2Apos counterparts. Conclusions: These results provide novel insights into the associations of NK phenotype with IL‐28B genotype and gene expression patterns, as well as the role of NKs in mediating IFN‐induced viral clearance of chronic HCV infection. (HEPATOLOGY 2011;)

Hepatitis C virus selectively perturbs the distal cholesterol synthesis pathway in a genotype-specific manner

Hepatitis C virus (HCV) subverts host cholesterol metabolism for key processes in its lifecycle. How this interference results in the frequently observed, genotype‐dependent clinical sequelae of hypocholesterolemia, hepatic steatosis, and insulin resistance (IR) remains incompletely understood. Hypocholesterolemia typically resolves after sustained viral response (SVR), implicating viral interference in host lipid metabolism. Using a targeted cholesterol metabolomic platform we evaluated paired HCV genotype 2 (G2) and G3 patient sera for changes in in vivo HCV sterol pathway metabolites. We compared HCV genotypic differences in baseline metabolites and following antiviral treatment to assess whether sterol perturbation resolved after HCV eradication. We linked these metabolites to IR and urine oxidative stress markers. In paired sera from HCV G2 (n = 13) and G3 (n = 20) patients, baseline sterol levels were lower in G3 than G2 for distal metabolites (7‐dehyrocholesterol (7DHC) 0.017 versus 0.023 mg/dL; Padj = 0.0524, cholesterol 140.9 versus 178.7 mg/dL; Padj = 0.0242) but not the proximal metabolite lanosterol. In HCV G3, SVR resulted in increased levels of distal metabolites (cholesterol [Δ55.2 mg/dL; Padj = 0.0015], 7DHC [Δ0.0075 mg/dL; Padj = 0.0026], lathosterol [Δ0.0430 mg/dL Padj = 0.0405]). In contrast, lanosterol was unchanged after SVR (P = 0.9515). Conclusion: HCV G3, but not G2, selectively interferes with the late cholesterol synthesis pathway, evidenced by lower distal sterol metabolites and preserved lanosterol levels. This distal interference resolves with SVR. Normal lanosterol levels provide a signal for the continued proteolysis of 3‐hydroxyl‐3‐methylglutaryl coenzyme A reductase, which may undermine other host responses to increase cholesterol synthesis. These data may provide a hypothesis to explain why hypocholesterolemia persists in chronic HCV infection, particularly in HCV G3, and is not overcome by host cholesterol compensatory mechanisms. (HEPATOLOGY 2012;56:49–56)

Genome-wide association study of interferon-related cytopenia in chronic hepatitis C patients

BACKGROUND & AIMS Interferon-alfa (IFN)-related cytopenias are common and may be dose-limiting. We performed a genome wide association study on a well-characterized genotype 1 HCV cohort to identify genetic determinants of peginterferon-α (pegIFN)-related thrombocytopenia, neutropenia, and leukopenia. METHODS 1604/3070 patients in the IDEAL study consented to genetic testing. Trial inclusion criteria included a platelet (Pl) count ≥80×10(9)/L and an absolute neutrophil count (ANC) ≥1500/mm(3). Samples were genotyped using the Illumina Human610-quad BeadChip. The primary analyses focused on the genetic determinants of quantitative change in cell counts (Pl, ANC, lymphocytes, monocytes, eosinophils, and basophils) at week 4 in patients >80% adherent to therapy (n=1294). RESULTS 6 SNPs on chromosome 20 were positively associated with Pl reduction (top SNP rs965469, p=10(-10)). These tag SNPs are in high linkage disequilibrium with 2 functional variants in the ITPA gene, rs1127354 and rs7270101, that cause ITPase deficiency and protect against ribavirin (RBV)-induced hemolytic anemia (HA). rs1127354 and rs7270101 showed strong independent associations with Pl reduction (p=10(-12), p=10(-7)) and entirely explained the genome-wide significant associations. We believe this is an example of an indirect genetic association due to a reactive thrombocytosis to RBV-induced anemia: Hb decline was inversely correlated with Pl reduction (r=-0.28, p=10(-17)) and Hb change largely attenuated the association between the ITPA variants and Pl reduction in regression models. No common genetic variants were associated with pegIFN-induced neutropenia or leucopenia. CONCLUSIONS Two ITPA variants were associated with thrombocytopenia; this was largely explained by a thrombocytotic response to RBV-induced HA attenuating IFN-related thrombocytopenia. No genetic determinants of pegIFN-induced neutropenia were identified.

Recurrence of hepatocellular carcinoma: importance of mRECIST response to chemoembolization and tumor size.

Determining risk for recurrence of hepatocellular carcinoma (HCC) following liver transplantation (LT) is an important clinical need. We assessed consecutive patients who underwent LT for HCC following sequential transarterial chemoembolization (TACE). Treatment response was assessed using modified response evaluation criteria in solid tumors (mRECIST) categories: complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). Cox proportional hazard models were used to predict HCC recurrence. One hundred seventy‐three patients underwent TACE and imaging to assess response prior to LT. TACE responses were: CR = 23.7%, PR = 24.3%, SD = 27.7% and PD = 24.3%. Five‐year HCC recurrence rate was 5.3% in patients responding to TACE (CR/PR), versus 17.6%, among patients who did not respond (SD/PD, p = 0.014). In multivariate analysis, independent pre‐LT predictors of recurrence were response to TACE and largest radiologic size of tumor (>3 cm vs. ≤3 cm). HCC recurrence rate for patients with tumor size >3 cm and no response to TACE was 35.8%, compared with 1.9% for patients with tumor size ≤3 cm and response to TACE (p = 0.0007). We conclude that mRECIST criteria and tumor size differentiate patients with high or low likelihood of HCC recurrence after LT. These findings raise the possibility of incorporating response to TACE and largest tumor size to identify patients at highest risk for HCC recurrence.

Inosine triphosphatase deficiency helps predict anaemia, anaemia management and response in chronic hepatitis C therapy

Anaemia frequently complicates peginterferon/ribavirin therapy for chronic hepatitis C infection. Better prediction of anaemia, ribavirin dose reduction or erythropoietin (EPO) need, may enhance patient management. Inosine triphosphatase (ITPA) genetic variants are associated with ribavirin‐induced anaemia and dose reduction; however, their impact in real‐life clinic patient cohorts remains to be defined. We studied 193 clinic patients with chronic hepatitis C infection of mixed viral genotype (genotype 1/4 n = 123, genotype 2/3, n = 70) treated with peginterferon/ribavirin. Patients were genotyped for ITPA polymorphisms rs1127354 and rs7270101 using Taqman primers. Hardy–Weinberg equilibrium was present. Estimated ITPA deficiency was graded on severity (0–3, no deficiency/mild/moderate/severe, n = 126/40/24/3, respectively). Multivariable models tested the association with anaemia at 4 weeks of treatment [including decline in haemoglobin (g/dL); haemoglobin <10 g/dL and haemoglobin decline >3 g/dL]; ribavirin dose reduction and EPO use and explored sustained viral response (SVR) to peginterferon/ribavirin. More severe ITPA deficiency was associated with less reduction in haemoglobin level (P < 0.001; R2 = 0.34), less ribavirin dose reduction (OR 0.42; (95% CI = 0.23–0.77); P = 0.005) and less EPO use [OR 0.53; (0.30–0.94); P = 0.029]. ITPA deficiency was associated with SVR [OR: 1.70; (1.02–2.83); P = 0.041] independently of clinical covariates (adjusted R2 = 0.31). In this clinical cohort, ITPA deficiency helped predict the risk of on‐treatment anaemia, ribavirin dose reduction, need for EPO support and was associated with SVR. For patients on HCV regimens including peginterferon/ribavirin, testing for ITPA deficiency may have clinical utility.

Host genomics and HCV treatment response

In 2009, an association between the interleukin‐28B (IL28B) polymorphism and treatment outcome for genotype 1 (G1) hepatitis C virus (HCV) infection, as well as spontaneous clearance of HCV, was reported. Since the initial publications, over 100 articles have appeared in the peer‐reviewed literature, with many more manuscripts in press and abstracts presented at scientific meetings. Despite the proliferation of data concerning the IL28B polymorphism and HCV infection, there remain many critical unanswered questions about clinical implications and the underlying biological mechanisms. In this review, we discuss the basic principles of genome‐wide association study methodologies that are important for interpreting the results of genetic association studies. We then review the current literature concerning the association between IL28B variants and interferon (IFN) treatment response in patients with chronic HCV infection, as well as spontaneous HCV clearance. We consider the relevance of the IL28B polymorphism to non‐G1 HCV, as well as the special treatment populations of HIV/HCV co‐infection and recurrent HCV post‐liver transplantation. We review current knowledge of the biological mechanisms underlying this genetic association, including the link to liver IFN‐stimulated gene expression, and identify continuing gaps in our knowledge and key research priorities. Finally, pegylated‐IFN and ribavirin is no longer the standard of care for the treatment of G1 HCV, and we conclude by considering the relevance of IL28B polymorphisms in the era of direct‐acting antivirals.