Much of the current data on nonalcoholic fatty liver disease (NAFLD) are derived from biopsy‐based studies that may introduce ascertainment and selection bias. Selection of patients for liver biopsy has implications for clinical practice and the reported epidemiology of NAFLD. The aim of this study was to determine patient factors predictive of histologic versus empiric clinical diagnosis of NAFLD in real‐world practice. Adults from TARGET‐NASH were included in this study. Descriptive statistics are provided for the cohort and compare the characteristics of histologic NAFLD versus patients with clinically diagnosed NAFLD, followed by logistic regression and machine‐learning models to describe predictors of liver biopsy. The records of 3,474 subjects were analyzed; median age was 59 years, 59% were female, 75% were White, and median body mass index was 32 kg/m 2 . Using histologic and/or clinical criteria, a diagnosis of nonalcoholic steatohepatitis was made in 37%, and cirrhosis in 33%. Comorbid conditions included cardiovascular disease (19%), mental health diagnoses (49%), and osteoarthritis (10%). Predictors of a biopsy diagnosis included White race, female sex, diabetes, and elevated alanine aminotransferase (ALT). ALT increased the odds of liver biopsy by 14% per 10‐point rise. Machine‐learning analyses showed non‐White patients with ALT <69 had only a 0.06 probability of undergoing liver biopsy. ALT was the dominant variable that determined liver biopsy. Conclusions : In this real‐world cohort of patients with NAFLD, two‐thirds of patients did not have a liver biopsy. These patients were more likely to be non‐White, older, with a normal ALT, showing potential gaps in or knowledge about this population.

Background: Evaluation of drug-induced serious hepatotoxicity (eDISH) is a tool used to assess and identify potential cases of drug-induced liver injury.eDISH was used to evaluate obeticholic acid (OCA) and placebo profiles in 2 double-blind (DB), placebo-controlled studies in patients with nonalcoholic steatohepatitis (NASH).FLINT was a 72-week study, which demonstrated statistically significant improvements in hepatocellular ballooning, steatosis, lobular inflammation, and fibrosis in patients treated with OCA compared to placebo.CONTROL was a 16-week study, which showed that the addition of low-dose atorvastatin reversed OCA-associated changes in LDL-C.The objective of this analysis was to use eDISH to determine if patients with NASH treated with OCA show increased markers of liver injury or whole liver dysfunction.Methods: eDISH methodology was applied to 278 patients treated with placebo (n=140) or 25mg OCA (n=138) from FLINT and 84 patients treated with placebo (n=21), 5mg OCA (n=20), 10mg OCA (n=21), or 25mg OCA (n=22) from CONTROL.Individual subject peak values of alanine aminotransferase (ALT) and total bilirubin throughout the DB treatment phase were plotted on an x-y chart as logarithm 10 values of multiples of elevations above the upper limit of the normal reference ranges (x ULN).Results: Overall, no OCA-treated patients were in the Hy's law quadrant (>3x ULN for ALT and >2x ULN for total bilirubin) compared with 1 placebo-treated patient in FLINT.The proportion of patients with peak ALT and total bilirubin values in the lower left quadrant (representing normal or near normal range) was higher in OCA-treated patients compared with placebo (FLINT: 91% OCA vs 84% placebo; CONTROL: 91% OCA vs 86% placebo).8% of OCA-treated patients from both FLINT and CONTROL presented in the Temple's corollary quadrant (>3x ULN for ALT and <2x ULN for total bilirubin) vs 14% (in both studies) for the placebo-treated patients.Across both studies (N=362), 4 patients were in the cholestasis quadrant (>2X ULN total bilirubin and <3X ULN for ALT); 1 placebotreated patient and 3 OCA-treated patients, including 1 patient with Gilbert's syndrome.Conclusions: In these 2 placebo-controlled, DB NASH studies, the eDISH analysis showed no trend for liver injury with OCA at doses up to and including 25 mg. Su1509

text will be published in the December 2018 issue of Hepatology and are also available for viewing on The Liver Meeting® app.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease. NAFLD comprises the spectrum from simple steatosis (nonalcoholic fatty liver, NAFL), to steatosis with inflammation (nonalcoholic steatohepatitis, NASH). Current primary therapy recommended for NAFLD is weight loss induced by lifestyle modification. The difficulty in achieving this has led to robust pharmacological therapy development. While new drugs may show efficacy in selected phase II/III clinical trial populations, their real-world effectiveness is unknown. TARGET-NASH is a 5-year, longitudinal, observational study of patients with NAFLD designed to evaluate the effectiveness of clinical practice interventions and provide practical information unobtainable in registration trials. A biological specimen repository is included in TARGET-NASH for translational studies of genomics and biomarkers of disease activity. Patients are enrolling at adult and pediatric sites representing multiple specialties. All patients being managed for NAFLD are eligible, whereas those in other NASH registries or clinical trials will be excluded. Enrolled patients range in age from 6 and up and will have 3years of clinical data reviewed. Patient comorbidities, concomitant medications, disease progression and off-label interventions will be assessed, and adverse outcomes, monitored. Confirming the use, safety and effectiveness of NAFLD interventions in children and adults and establishing pragmatic methods of assessing disease progression under real-world conditions are key study outcomes. Ultimately, TARGET-NASH will establish a large, diverse registry of NAFLD patients at academic and community practices to be leveraged to improve health and reduce development of cirrhosis and hepatocellular carcinoma.

Background Many patients with primary biliary cholangitis have an inadequate response to first-line therapy with ursodeoxycholic acid. Seladelpar is a potent, selective agonist for the peroxisome proliferator-activated receptor-delta (PPAR-d), which is implicated in bile acid homoeostasis. This first-in-class study evaluated the anti-cholestatic effects and safety of seladelpar in patients with an inadequate response to ursodeoxycholic acid.Methods The study was a 12-week, double-blind, placebo-controlled, phase 2 trial of patients with alkaline phosphatase of at least 1.67 times the upper limit of normal (ULN) despite treatment with ursodeoxycholic acid. Patients, recruited at 29 sites in North America and Europe, were randomly assigned to placebo, seladelpar 50 mg/day, or seladelpar 200 mg/day while ursodeoxycholic acid was continued. Randomisation was done centrally (1:1:1) by a computerised system using an interactive voice-web response system with a block size of three. Randomisation was stratified by region (North America and Europe). The primary outcome was the percentage change from baseline in alkaline phosphatase over 12 weeks, analysed in the modified intention-to-treat (ITT) population (any randomised patient who received at least one dose of medication and had at least one post-baseline alkaline phosphatase evaluation). This study is registered with ClinicalTrials. gov (NCT02609048) and the EU Clinical Trials Registry (EudraCT2015-002698-39).Findings Between Nov 4, 2015, and May 26, 2016, 70 patients were screened at 29 sites in North America and Europe. During recruitment, three patients treated with seladelpar developed fully reversible, asymptomatic grade 3 alanine aminotransferase increases (one on 50 mg, two on 200 mg), ranging from just over five to 20 times the ULN; as a result, the study was terminated after 41 patients were randomly assigned. The modified ITT population consisted of 12 patients in the placebo group, 13 in the seladelpar 50 mg group, and 10 in the seladelpar 200 mg group. Mean changes from baseline in alkaline phosphatase were -2% (SD 16) in the placebo group, -53% (14) in the seladelpar 50 mg group, and -63% (8) in the seladelpar 200 mg group. Changes in both seladelpar groups versus placebo were significant (p<0.0001 for both groups vs placebo), with no significant difference between the two seladelpar groups (p=0.1729). All five patients who received seladelpar for 12 weeks had normal alkaline phosphatase values at the end of treatment, based on a central laboratory ULN for alkaline phosphatase of 116 U/L. The most frequently reported adverse events were pruritus (16%; one patient on placebo, four on seladelpar 50 mg, and one on seladelpar 200 mg), nausea (13%; one patient on placebo, three on seladelpar 50 mg, and one on seladelpar 200 mg), diarrhoea (10%; two patients on placebo, one on seladelpar 50 mg, and one on seladelpar 200 mg), dyspepsia (8%; two patients on seladelpar 50 mg and one on seladelpar 200 mg), muscle spasms (8%; three patients on seladelpar 200 mg), myalgia (8%; one patient on placebo and two on seladelpar 200 mg), and dizziness (8%; one patient on placebo and two on seladelpar 50 mg).Interpretation Seladelpar normalised alkaline phosphatase levels in patients who completed 12 weeks of treatment. However, treatment was associated with grade 3 increases in aminotransferases and the study was stopped early. The effects of seladelpar should be explored at lower doses.

Effective antiviral therapy is essential for achieving sustained virological response (SVR) in hepatitis C virus (HCV)‐infected patients. The phase 2 COSMOS study reported high SVR rates in treatment‐naive and prior null‐responder HCV genotype (GT) 1‐infected patients receiving simeprevir+sofosbuvir±ribavirin for 12 or 24 weeks. OPTIMIST‐1 (NCT02114177) was a multicenter, randomized, open‐label study assessing the efficacy and safety of 12 and 8 weeks of simeprevir+sofosbuvir in HCV GT1‐infected treatment‐naive and treatment‐experienced patients without cirrhosis. Patients were randomly assigned (1:1; stratified by HCV GT/subtype and presence or absence of NS3 Q80K polymorphism [GT1b, GT1a with Q80K, GT1a without Q80K]), prior HCV treatment history, and IL28B GT [CC, non‐CC]) to simeprevir 150 mg once daily+sofosbuvir 400 mg once daily for 12 or 8 weeks. The primary efficacy endpoint was SVR rate 12 weeks after end of treatment (SVR12). Superiority in SVR12 was assessed for simeprevir+sofosbuvir at 12 and 8 weeks versus a composite historical control SVR rate. Enrolled were 310 patients, who were randomized and received treatment (n = 155 in each arm). SVR12 with simeprevir+sofosbuvir for 12 weeks (97% [150/155; 95% confidence interval 94%‐100%]) was superior to the historical control (87%). SVR12 with simeprevir+sofosbuvir for 8 weeks (83% [128/155; 95% confidence interval 76‐89%]) was not superior to the historical control (83%). The most frequent adverse events were nausea, headache, and fatigue (12‐week arm: 15% [23/155], 14% [22/155], and 12% [19/155]; 8‐week arm: 9% [14/155], 17% [26/155], and 15% [23/155], respectively). No patients discontinued treatment due to an adverse event. One (1%, 12‐week arm) and three (2%, 8‐week arm) patients experienced a serious adverse event (all unrelated to study treatment). Conclusion: Simeprevir+sofosbuvir for 12 weeks is highly effective in the treatment of HCV GT1‐infected patients without cirrhosis, including those with Q80K. (Hepatology 2016;64:370‐380)

BACKGROUND AND AIMS:Many currently available direct-acting antiviral (DAA) regimens are less effective against HCV genotype 3 than against other HCV genotypes. The all-oral, pangenotypic DAA combination of daclatasvir (NS5A inhibitor) + sofosbuvir (nucleotide NS5B inhibitor) was studied in genotype 3-infected treatment-naive and -experienced patients (ALLY-3) who achieved rates of sustained virological response at post-treatment Week 12 (SVR12) of 90 and 86% respectively. In this analysis, we assessed whether on-treatment responses to daclatasvir + sofosbuvir in genotype 3-infected patients could predict treatment outcome.METHODS:In ALLY-3, treatment-naive and -experienced patients, with or without cirrhosis, were treated with daclatasvir + sofosbuvir for 12 weeks. HCV RNA kinetics and categorical virological responses on treatment were assessed. The proportions of responders and nonresponders by study week, and time to first undetectable HCV RNA, were analysed for utility in predicting treatment outcome.RESULTS:Overall, HCV RNA levels declined rapidly during Week 1 of treatment in both treatment-naive and -experienced cohorts. Although patients with cirrhosis had a slower initial virological response as measured by the proportion of patients with HCV RNA below the lower limit of quantification at Week 1, responses converged thereafter. Positive and negative predictive values calculated for on-treatment responses were generally comparable with the overall SVR12 rate and were therefore limited indicators of outcome. SVR12 rates were not impacted by time to first undetectable HCV RNA.CONCLUSIONS:On-treatment responses are not useful predictors of ultimate virological response to the daclatasvir + sofosbuvir regimen.

Optimism for patients with genotype 4 HCV infection: Clinical trials with direct-acting antivirals finally availableJournal of HepatologyVol. 62Issue 5PreviewThis Editorial discusses three recent original papers related to direct-acting antivirals (DAAs) for the treatment of chronic genotype (GT) 4 HCV infection, published in this issue of the Journal of Hepatology [1–3]. Full-Text PDF While most hepatitis C virus (HCV) treatment studies have focused on genotypes 1–3, genotype 4 studies are lacking, even though it is common in the Middle East and Africa and has a growing prevalence elsewhere [1Sievert W. Altraif I. Razavi H.A. Abdo A. Ahmed E.A. Alomair A. et al.A systematic review of hepatitis C virus epidemiology in Asia, Australia and Egypt.Liver Int. 2011; 31: 61-80Crossref PubMed Scopus (423) Google Scholar, 2Karoney M.J. Siika A.M. Hepatitis C virus (HCV) infection in Africa: a review.Pan Afr Med J. 2013; 14: 44PubMed Google Scholar]. The most effective genotype 4 treatment currently approved is peginterferon (PegIFN)/ribavirin with sofosbuvir. However, both PegIFN and ribavirin carry a significant tolerability burden, and there remains a need for effective and better tolerated all-oral combinations for genotype 4 treatment.The combination of daclatasvir (NS5A inhibitor), asunaprevir (NS3 protease inhibitor), and beclabuvir (BMS-791325) – a non-nucleoside NS5B polymerase inhibitor with comparable activity against genotypes 1a, 1b, 3a, 4a, and 5a (50% effective concentration 3–18 nM) and more variable activity against genotype 6a (9–125 nM) [[3]Lemm J.A. Liu M. Gentles R.G. Ding M. Voss S. Pelosi L.A. et al.Preclinical characterization of BMS-791325, an allosteric Inhibitor of hepatitis C virus NS5B polymerase.Antimicrob Agents Chemother. 2014; 58: 3485-3495Crossref PubMed Scopus (51) Google Scholar] – has demonstrated sustained virologic response (SVR) rates >90% in treatment-naïve genotype 1 infection [[4]Everson G.T. Sims K.D. Rodriguez-Torres M. Hezode C. Lawitz E. Bourliere M. et al.Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.Gastroenterology. 2014; 146: 420-429Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar]. Each of these agents is active in vitro against genotype 4. We therefore undertook a randomized, open-label, phase 2a exploratory evaluation of this regimen in treatment-naïve patients with genotype 4 infection.The patients (n = 21) were enrolled at nine sites in the USA as an expansion of a larger study (ClinicalTrials.gov number NCT01455090) where the design and methods have been fully described elsewhere [[4]Everson G.T. Sims K.D. Rodriguez-Torres M. Hezode C. Lawitz E. Bourliere M. et al.Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.Gastroenterology. 2014; 146: 420-429Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar]. Briefly, treatment-naïve adults with chronic genotype 4 HCV infection were randomized 1:1 to receive a twice-daily oral regimen comprising of 75 mg or 150 mg of beclabuvir, each with daclatasvir (30 mg) and asunaprevir (200 mg), for 12 weeks with 48 weeks of post-treatment follow-up. The primary endpoint was SVR at post-treatment Week 12 (SVR12), defined as HCV RNA below 25 IU/ml (COBAS TaqMan V2 assay; Roche Molecular Diagnostics, Pleasanton, CA, USA), with or without target detected. In the primary modified intention-to-treat analysis, missing HCV RNA data at post-treatment Week 12 was considered failure. An imputed analysis with missing Week 12 SVR data backwards-imputed from the next available RNA measurement was also performed. Sample size was not derived on the basis of formal statistical power. At an assumed population SVR12 of 90%, 12 patients per arm was predicted to yield an 11% probability of underestimating – and a 28% probability of overestimating – SVR12 by ⩾10%, with a 72% probability of observing a safety event with an incidence of 10%. Patients with compensated cirrhosis were permitted although none were enrolled.Viral subtype was assessed by phylogenetic alignment. Baseline NS5A amino acid sequences (positions 9–213) were aligned with 39 genotype 4 sequences from the European HCV Database (http://euhcvdb.ibcp.fr/euHCVdb/) using the ClustalW algorithm in the AlignX program of Vector NTI (Invitrogen, Carlsbad, CA, USA). Neighbor-joining trees (1000 bootstrap replicates) were generated from these alignments using Mega version 5.05 and were rooted on the ED43 genotype 4 reference sequence (Genbank accession number Y11604).Resistance-associated polymorphisms (RAPs) associated with reduced susceptibility to daclatasvir (e.g. NS5A-L28, -L30, -Y93 variants), beclabuvir (e.g. NS5B-P495 variants) or asunaprevir (e.g. NS3-Q80, -D168, -R155 variants) were determined at baseline by population-based sequencing.Baseline characteristics were comparable between treatment arms and are shown in Supplementary Table 1. Overall patients were white and male (91% and 62% respectively), with a mean age of 51 years. Ethnic ancestry was not collected for study entry, but post hoc enquiries established 8/21 patients (38%) to be of Near or Middle Eastern origin (5 Egyptian, 1 Saudi, 1 Afghan, 1 not specified). All 21 patients completed randomized therapy and adherence to study medication was high, with 90% (19/21) assessed as ⩾95% adherent to all three study drugs, assessed by pill counts and dosing diaries. There were no virologic failures or post-treatment rebounds in either treatment arm. HCV RNA decline was rapid: median (range) log10 change from baseline at day 7 was −4.39 (−4.91, −2.95) IU/ml in the 75 mg beclabuvir group and −4.01 (−5.03, −3.47) IU/ml in the 150 mg group. All patients had <25 IU/ml by Week 2 on-treatment. Two patients, one in each treatment arm, were missing data at post-treatment Week 12 but were confirmed to have <25 IU/ml of HCV RNA at their next attended visits at post-treatment Weeks 24 and/or 36. SVR12 was therefore achieved by 90.9% (10/11) of patients receiving beclabuvir 75 mg and by 90.0% (9/10) patients receiving 150 mg in the primary mITT analysis, and by 100% of patients in both arms by the imputed analysis (Table 1). Concordance between SVR12 and SVR24 was 100% in the 9 patients in each arm with available HCV RNA data at both these post-treatment time points.Table 1Virologic responses.aThree patients in the beclabuvir 75 mg and one patient in the 150 mg group had missing data at post-treatment Week 4; all four patients achieved SVR12, SVR24, or SVR36.bOne patient in each group had missing data at post-treatment Week 12, but was established to have an SVR result at their next visits (SVR24 and SVR36). Open table in a new tab There were no serious adverse events, discontinuations for adverse events, or grade 3–4 adverse events in the study. Fifteen patients (71%) experienced adverse mild-moderate events, seven patients in the 75 mg arm and eight in the 150 mg arm; all events were of mild (11/15 patients) or moderate (4/15) intensity. Only one grade 3 laboratory abnormality was observed: an isolated incident of hypophosphatemia (1.9 mg/dl; lower limit of normal = 2.4 mg/dl) was noted at the end of treatment/Week 12 visit and normalized by post-treatment Week 4. No grade 3 or 4 transaminase or bilirubin elevations were observed, and there was no evidence of beclabuvir dose-related differences in the regimen safety profile. Headache, insomnia, nausea and pain were reported in more than 10% of patients (Supplementary Table 2).In the phylogenetic analysis, virus from most patients (n = 16) grouped with genotype 4a reference sequences, with the remainder grouping with 4l (n = 2), 4d, 4n or 4o (n = 1 each; Supplementary Fig. 1). No baseline RAPs in NS3 or NS5B were observed. Twelve NS5A RAPs at positions 28, 30 and/or 93 were identified in nine patients: L28M (n = 2), L30R (n = 4), L30H (n = 1), L28M + L30A (n = 1) and L28L/M + L30L/H + Y93Y/C (n = 1). In a previous study, L28 and L30 substitutions emerged together in genotype 4 infected patients with virologic failure who had received daclatasvir combined with PegIFNα and ribavirin [[5]McPhee F. Zhou N. Ueland J. Yu F. Yang X. Monikowski A. et al.Pre-existence, emergence, and persistence of HCV genotype 4 NS5A resistance variants from the phase 2b COMMAND-1 study: daclatasvir plus peginterferon-alfa /ribavirin in treatment-naïve patients [abstract].J Hepatol. 2013; 58: S409Abstract Full Text PDF PubMed Google Scholar]. A further 22 NS5A polymorphisms were observed at positions 54, 58, and 62; polymorphisms at these positions are associated with minimal changes in daclatasvir susceptibility in replicon assays (<2-fold elevation in EC50). Most patient samples (n = 19) had a D62E substitution relative to the ED43 reference strain. Other minor polymorphisms were D62N (n = 1), P58T (n = 1) and P58S (n = 1). These RAPs did not affect virologic response in this study and all patients achieved SVR.Thus, in this exploratory study, 12 weeks of twice-daily therapy with daclatasvir, asunaprevir, and beclabuvir (75 mg or 150 mg) resulted in a rapid and sustained decline in HCV viraemia in treatment-naïve patients with genotype 4 infection, with SVR12 rates similar to the 96% SVR12 seen in genotype 4 after 12 weeks of sofosbuvir plus PegIFN/ribavirin [[6]Food and Drug Administration. SOVALDI™ (Sofosbuvir) Prescribing Information; 2014.Google Scholar]. There were no notable safety or tolerability issues, consistent with previous data for this regimen with genotype 1 [[7]Everson GT, Sims KD, Thuluvath PJ, Lawitz E, Hassanein T, Rodriguez-Torrez M et al. Phase 2b study of the interferon-free and ribavirin-free combination of daclatasvir, asunaprevir, and BMS-791325 for 12 weeks in treatment-naive patients with chronic HCV genotype 1 infection. In: 64th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2013) Washington, DC, November 1–5, 2013. Abstract LB-1.Google Scholar]. Thus, this combination of oral agents for treatment of genotype 4 may potentially provide an improved safety and tolerability profile compared with current PegIFN/ribavirin-containing regimens. It may also offer a tolerability advantage compared with several ribavirin-containing all-oral regimens for genotype 4 which are currently under investigation [8Ruane P.J. Ain D. Stryker R. Meshrekey R. Soliman M. Wolfe P.R. et al.Sofosbuvir plus ribavirin for the treatment of chronic genotype 4 hepatitis C virus infection in patients of Egyptian ancestry.J Hepatol. 2015; 62: 1040-1046Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9Hezode C. Marcellin P. Pol S. Hassanein T. Fleischer-Stepniewska K. Baykal T. et al.Results from the phase 2 PEARL-I study: interferon-free regimens of ABT-450/r + ABT-267 with or without ribavirin in patients with HCV genotype 4 infection.J Hepatol. 2014; 60: S24Abstract Full Text PDF Google Scholar, 10Lawitz E. Rodriguez-Torres M. Nguyen T. Sheikh A. Tobias H. Galati J. et al.A phase II study of samatasvir (IDX719) in combination with simeprevir and ribavirin in treatment-naïve HCV-infected subjects with genotypes 1b and 4 (HELIX-1 study).J Hepatol. 2014; 60: S495-S496Abstract Full Text PDF Google Scholar]. Adherence to the three components of the regimen (supplied in separate bottles) was high, and may be further improved by the use of a fixed-dose combination tablet currently under phase 3 evaluation.Although baseline RAPs in NS5A were observed in just under half the patients assessed, there were no on-treatment virologic failures, and no post-treatment relapses through the primary endpoint or in any patient for whom post-SVR12 data were available at time of analysis. A combination of three different mechanistic classes of direct-acting agent may conceivably reduce the virological impact of reduced susceptibility to any one of them, but the overall impact of baseline RAPs on response to this regimen will require evaluation in a larger dataset.The primary limitations of this study are its small sample size and the relatively mild stage of liver disease presented, both of which limit the extent to which study outcomes can be extrapolated to the broader virus and patient populations. Coupled with the present results, ongoing phase 3 studies in genotype 1 infection will help inform future studies in patients with genotype 4. Most infections were with genotype 4a, and although the consistent virologic results in the 24% of patients with non-4a infections suggest that this regimen will be effective across subtypes, a larger study in a more diverse range of genotype 4 infections will be needed to confirm this. In addition, there were no cirrhotic patients and only four patients with a FibroTest-derived fibrosis staging above F2 (FibroTest score >0.58). The absence of patients with advanced liver disease precludes conclusions about safety or efficacy in this population, although it is relevant that SVR rates above 90% have subsequently been observed for this regimen ± ribavirin in genotype 1 infected, treatment-naïve patients with compensated cirrhosis in the phase 3 UNITY-2 study [[11]Muir A, Poordad F, Lalezari JP, Everson GT, Dore GJ, Kwo P et al. All-oral fixed-dose combination therapy with daclatasvir/asunaprevir/BMS-791325, +/− ribavirin, for patients with chronic HCV genotype 1 infection and compensated cirrhosis: UNITY-2 Phase 3 SVR12. In: 65th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2014), Boston, MA, November 7–12, 2014. Abstract LB-2.Google Scholar]. The sample size is also insufficient for detection of low-frequency safety-related events; however, this limitation is mitigated to some extent by the similarly favorable safety profile observed with this regimen in larger populations of patients with genotype 1 infection [7Everson GT, Sims KD, Thuluvath PJ, Lawitz E, Hassanein T, Rodriguez-Torrez M et al. Phase 2b study of the interferon-free and ribavirin-free combination of daclatasvir, asunaprevir, and BMS-791325 for 12 weeks in treatment-naive patients with chronic HCV genotype 1 infection. In: 64th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2013) Washington, DC, November 1–5, 2013. Abstract LB-1.Google Scholar, 11Muir A, Poordad F, Lalezari JP, Everson GT, Dore GJ, Kwo P et al. All-oral fixed-dose combination therapy with daclatasvir/asunaprevir/BMS-791325, +/− ribavirin, for patients with chronic HCV genotype 1 infection and compensated cirrhosis: UNITY-2 Phase 3 SVR12. In: 65th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2014), Boston, MA, November 7–12, 2014. Abstract LB-2.Google Scholar, 12Poordad F, Sievert W, Mollison L, Brau N, Levin J, Sepe T et al. All-oral fixed-dose combination therapy with daclatasvir/asunaprevir/BMS-791325 for non-cirrhotic patients with HCV genotype 1 infection: UNITY-1 Phase 3 SVR12 results. 65th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2014), Boston, MA, November 7–12, 2014. Abstract LB-7.Google Scholar].In summary, in this exploratory evaluation, 12 weeks of treatment with the IFN- and ribavirin-free combination of daclatasvir, asunaprevir and beclabuvir was well tolerated and resulted in a high rate of SVR in previously untreated patients with chronic HCV genotype 4 infection, consistent with previous results in genotype 1.Conflict of interestT. Hassanein has received consulting fees from Bristol-Myers Squibb. E. Lawitz has received research grants from AbbVie, Achillion, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Idenix, Janssen, Merck, Novartis, Presidio, Roche, Santaris, and Vertex, and consulting fees and/or non-financial support from AbbVie, Achillion, BioCryst, Biotica, Enanta, Gilead Sciences, Idenix, Janssen, Kadmon, Merck, Novartis, Presidio, Santaris, Theravance, and Vertex. L. Webster has received consulting fees from AcelRx, Acura, AstraZeneca, BioDelivery Sciences International, Boehringer Ingelheim, Boston Scientific, and Collegium. Z. Younossi has received consulting fees from Salix, Janssen, Vertex, Gilead Sciences, Enterome, and Coneatus. P.J. Thuluvath has received research grant support from Bristol-Myers Squibb, Gilead Sciences, Vertex, Isai, AbbVie, and Salix, and speaker fees from Gilead Sciences, Onyx, AbbVie, and Bayer. K.D. Sims, B. Rege, N. Zhou, M. Wind-Rotolo, E. Chung, and D.M. Grasela are employees and shareholders of Bristol-Myers Squibb. H. Zhou, F. McPhee, A. Griffies, and D.F. Gardiner are employees of Bristol-Myers Squibb. M. Bennett, N. Gitlin, T. Nguyen, and H. Schwartz have no conflicts of interest to disclose.Financial supportThis study was entirely funded by the sponsor, Bristol-Myers Squibb. Editorial assistance with the manuscript was provided by Richard Boehme, PhD, and Nick Fitch, PhD, of Articulate Science, with funding from the sponsor.Authors' contributionsK.D. Sims, D.M. Grasela, E. Chung, A. Griffies, and D.F. Gardiner designed the study. T. Hassanein, M. Bennett, N. Gitlin, E. Lawitz, T. Nguyen, L. Webster, Z. Younossi, H. Schwartz, and P.J. Thuluvath recruited patients and obtained data. H. Zhou, B. Rege, F. McPhee, N. Zhou, and M. Wind-Rotolo analyzed the data. All authors interpreted the data, participated in writing the manuscript, and approved the final version of the manuscript. While most hepatitis C virus (HCV) treatment studies have focused on genotypes 1–3, genotype 4 studies are lacking, even though it is common in the Middle East and Africa and has a growing prevalence elsewhere [1Sievert W. Altraif I. Razavi H.A. Abdo A. Ahmed E.A. Alomair A. et al.A systematic review of hepatitis C virus epidemiology in Asia, Australia and Egypt.Liver Int. 2011; 31: 61-80Crossref PubMed Scopus (423) Google Scholar, 2Karoney M.J. Siika A.M. Hepatitis C virus (HCV) infection in Africa: a review.Pan Afr Med J. 2013; 14: 44PubMed Google Scholar]. The most effective genotype 4 treatment currently approved is peginterferon (PegIFN)/ribavirin with sofosbuvir. However, both PegIFN and ribavirin carry a significant tolerability burden, and there remains a need for effective and better tolerated all-oral combinations for genotype 4 treatment. The combination of daclatasvir (NS5A inhibitor), asunaprevir (NS3 protease inhibitor), and beclabuvir (BMS-791325) – a non-nucleoside NS5B polymerase inhibitor with comparable activity against genotypes 1a, 1b, 3a, 4a, and 5a (50% effective concentration 3–18 nM) and more variable activity against genotype 6a (9–125 nM) [[3]Lemm J.A. Liu M. Gentles R.G. Ding M. Voss S. Pelosi L.A. et al.Preclinical characterization of BMS-791325, an allosteric Inhibitor of hepatitis C virus NS5B polymerase.Antimicrob Agents Chemother. 2014; 58: 3485-3495Crossref PubMed Scopus (51) Google Scholar] – has demonstrated sustained virologic response (SVR) rates >90% in treatment-naïve genotype 1 infection [[4]Everson G.T. Sims K.D. Rodriguez-Torres M. Hezode C. Lawitz E. Bourliere M. et al.Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.Gastroenterology. 2014; 146: 420-429Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar]. Each of these agents is active in vitro against genotype 4. We therefore undertook a randomized, open-label, phase 2a exploratory evaluation of this regimen in treatment-naïve patients with genotype 4 infection. The patients (n = 21) were enrolled at nine sites in the USA as an expansion of a larger study (ClinicalTrials.gov number NCT01455090) where the design and methods have been fully described elsewhere [[4]Everson G.T. Sims K.D. Rodriguez-Torres M. Hezode C. Lawitz E. Bourliere M. et al.Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.Gastroenterology. 2014; 146: 420-429Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar]. Briefly, treatment-naïve adults with chronic genotype 4 HCV infection were randomized 1:1 to receive a twice-daily oral regimen comprising of 75 mg or 150 mg of beclabuvir, each with daclatasvir (30 mg) and asunaprevir (200 mg), for 12 weeks with 48 weeks of post-treatment follow-up. The primary endpoint was SVR at post-treatment Week 12 (SVR12), defined as HCV RNA below 25 IU/ml (COBAS TaqMan V2 assay; Roche Molecular Diagnostics, Pleasanton, CA, USA), with or without target detected. In the primary modified intention-to-treat analysis, missing HCV RNA data at post-treatment Week 12 was considered failure. An imputed analysis with missing Week 12 SVR data backwards-imputed from the next available RNA measurement was also performed. Sample size was not derived on the basis of formal statistical power. At an assumed population SVR12 of 90%, 12 patients per arm was predicted to yield an 11% probability of underestimating – and a 28% probability of overestimating – SVR12 by ⩾10%, with a 72% probability of observing a safety event with an incidence of 10%. Patients with compensated cirrhosis were permitted although none were enrolled. Viral subtype was assessed by phylogenetic alignment. Baseline NS5A amino acid sequences (positions 9–213) were aligned with 39 genotype 4 sequences from the European HCV Database (http://euhcvdb.ibcp.fr/euHCVdb/) using the ClustalW algorithm in the AlignX program of Vector NTI (Invitrogen, Carlsbad, CA, USA). Neighbor-joining trees (1000 bootstrap replicates) were generated from these alignments using Mega version 5.05 and were rooted on the ED43 genotype 4 reference sequence (Genbank accession number Y11604). Resistance-associated polymorphisms (RAPs) associated with reduced susceptibility to daclatasvir (e.g. NS5A-L28, -L30, -Y93 variants), beclabuvir (e.g. NS5B-P495 variants) or asunaprevir (e.g. NS3-Q80, -D168, -R155 variants) were determined at baseline by population-based sequencing. Baseline characteristics were comparable between treatment arms and are shown in Supplementary Table 1. Overall patients were white and male (91% and 62% respectively), with a mean age of 51 years. Ethnic ancestry was not collected for study entry, but post hoc enquiries established 8/21 patients (38%) to be of Near or Middle Eastern origin (5 Egyptian, 1 Saudi, 1 Afghan, 1 not specified). All 21 patients completed randomized therapy and adherence to study medication was high, with 90% (19/21) assessed as ⩾95% adherent to all three study drugs, assessed by pill counts and dosing diaries. There were no virologic failures or post-treatment rebounds in either treatment arm. HCV RNA decline was rapid: median (range) log10 change from baseline at day 7 was −4.39 (−4.91, −2.95) IU/ml in the 75 mg beclabuvir group and −4.01 (−5.03, −3.47) IU/ml in the 150 mg group. All patients had <25 IU/ml by Week 2 on-treatment. Two patients, one in each treatment arm, were missing data at post-treatment Week 12 but were confirmed to have <25 IU/ml of HCV RNA at their next attended visits at post-treatment Weeks 24 and/or 36. SVR12 was therefore achieved by 90.9% (10/11) of patients receiving beclabuvir 75 mg and by 90.0% (9/10) patients receiving 150 mg in the primary mITT analysis, and by 100% of patients in both arms by the imputed analysis (Table 1). Concordance between SVR12 and SVR24 was 100% in the 9 patients in each arm with available HCV RNA data at both these post-treatment time points. aThree patients in the beclabuvir 75 mg and one patient in the 150 mg group had missing data at post-treatment Week 4; all four patients achieved SVR12, SVR24, or SVR36. bOne patient in each group had missing data at post-treatment Week 12, but was established to have an SVR result at their next visits (SVR24 and SVR36). There were no serious adverse events, discontinuations for adverse events, or grade 3–4 adverse events in the study. Fifteen patients (71%) experienced adverse mild-moderate events, seven patients in the 75 mg arm and eight in the 150 mg arm; all events were of mild (11/15 patients) or moderate (4/15) intensity. Only one grade 3 laboratory abnormality was observed: an isolated incident of hypophosphatemia (1.9 mg/dl; lower limit of normal = 2.4 mg/dl) was noted at the end of treatment/Week 12 visit and normalized by post-treatment Week 4. No grade 3 or 4 transaminase or bilirubin elevations were observed, and there was no evidence of beclabuvir dose-related differences in the regimen safety profile. Headache, insomnia, nausea and pain were reported in more than 10% of patients (Supplementary Table 2). In the phylogenetic analysis, virus from most patients (n = 16) grouped with genotype 4a reference sequences, with the remainder grouping with 4l (n = 2), 4d, 4n or 4o (n = 1 each; Supplementary Fig. 1). No baseline RAPs in NS3 or NS5B were observed. Twelve NS5A RAPs at positions 28, 30 and/or 93 were identified in nine patients: L28M (n = 2), L30R (n = 4), L30H (n = 1), L28M + L30A (n = 1) and L28L/M + L30L/H + Y93Y/C (n = 1). In a previous study, L28 and L30 substitutions emerged together in genotype 4 infected patients with virologic failure who had received daclatasvir combined with PegIFNα and ribavirin [[5]McPhee F. Zhou N. Ueland J. Yu F. Yang X. Monikowski A. et al.Pre-existence, emergence, and persistence of HCV genotype 4 NS5A resistance variants from the phase 2b COMMAND-1 study: daclatasvir plus peginterferon-alfa /ribavirin in treatment-naïve patients [abstract].J Hepatol. 2013; 58: S409Abstract Full Text PDF PubMed Google Scholar]. A further 22 NS5A polymorphisms were observed at positions 54, 58, and 62; polymorphisms at these positions are associated with minimal changes in daclatasvir susceptibility in replicon assays (<2-fold elevation in EC50). Most patient samples (n = 19) had a D62E substitution relative to the ED43 reference strain. Other minor polymorphisms were D62N (n = 1), P58T (n = 1) and P58S (n = 1). These RAPs did not affect virologic response in this study and all patients achieved SVR. Thus, in this exploratory study, 12 weeks of twice-daily therapy with daclatasvir, asunaprevir, and beclabuvir (75 mg or 150 mg) resulted in a rapid and sustained decline in HCV viraemia in treatment-naïve patients with genotype 4 infection, with SVR12 rates similar to the 96% SVR12 seen in genotype 4 after 12 weeks of sofosbuvir plus PegIFN/ribavirin [[6]Food and Drug Administration. SOVALDI™ (Sofosbuvir) Prescribing Information; 2014.Google Scholar]. There were no notable safety or tolerability issues, consistent with previous data for this regimen with genotype 1 [[7]Everson GT, Sims KD, Thuluvath PJ, Lawitz E, Hassanein T, Rodriguez-Torrez M et al. Phase 2b study of the interferon-free and ribavirin-free combination of daclatasvir, asunaprevir, and BMS-791325 for 12 weeks in treatment-naive patients with chronic HCV genotype 1 infection. In: 64th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2013) Washington, DC, November 1–5, 2013. Abstract LB-1.Google Scholar]. Thus, this combination of oral agents for treatment of genotype 4 may potentially provide an improved safety and tolerability profile compared with current PegIFN/ribavirin-containing regimens. It may also offer a tolerability advantage compared with several ribavirin-containing all-oral regimens for genotype 4 which are currently under investigation [8Ruane P.J. Ain D. Stryker R. Meshrekey R. Soliman M. Wolfe P.R. et al.Sofosbuvir plus ribavirin for the treatment of chronic genotype 4 hepatitis C virus infection in patients of Egyptian ancestry.J Hepatol. 2015; 62: 1040-1046Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9Hezode C. Marcellin P. Pol S. Hassanein T. Fleischer-Stepniewska K. Baykal T. et al.Results from the phase 2 PEARL-I study: interferon-free regimens of ABT-450/r + ABT-267 with or without ribavirin in patients with HCV genotype 4 infection.J Hepatol. 2014; 60: S24Abstract Full Text PDF Google Scholar, 10Lawitz E. Rodriguez-Torres M. Nguyen T. Sheikh A. Tobias H. Galati J. et al.A phase II study of samatasvir (IDX719) in combination with simeprevir and ribavirin in treatment-naïve HCV-infected subjects with genotypes 1b and 4 (HELIX-1 study).J Hepatol. 2014; 60: S495-S496Abstract Full Text PDF Google Scholar]. Adherence to the three components of the regimen (supplied in separate bottles) was high, and may be further improved by the use of a fixed-dose combination tablet currently under phase 3 evaluation. Although baseline RAPs in NS5A were observed in just under half the patients assessed, there were no on-treatment virologic failures, and no post-treatment relapses through the primary endpoint or in any patient for whom post-SVR12 data were available at time of analysis. A combination of three different mechanistic classes of direct-acting agent may conceivably reduce the virological impact of reduced susceptibility to any one of them, but the overall impact of baseline RAPs on response to this regimen will require evaluation in a larger dataset. The primary limitations of this study are its small sample size and the relatively mild stage of liver disease presented, both of which limit the extent to which study outcomes can be extrapolated to the broader virus and patient populations. Coupled with the present results, ongoing phase 3 studies in genotype 1 infection will help inform future studies in patients with genotype 4. Most infections were with genotype 4a, and although the consistent virologic results in the 24% of patients with non-4a infections suggest that this regimen will be effective across subtypes, a larger study in a more diverse range of genotype 4 infections will be needed to confirm this. In addition, there were no cirrhotic patients and only four patients with a FibroTest-derived fibrosis staging above F2 (FibroTest score >0.58). The absence of patients with advanced liver disease precludes conclusions about safety or efficacy in this population, although it is relevant that SVR rates above 90% have subsequently been observed for this regimen ± ribavirin in genotype 1 infected, treatment-naïve patients with compensated cirrhosis in the phase 3 UNITY-2 study [[11]Muir A, Poordad F, Lalezari JP, Everson GT, Dore GJ, Kwo P et al. All-oral fixed-dose combination therapy with daclatasvir/asunaprevir/BMS-791325, +/− ribavirin, for patients with chronic HCV genotype 1 infection and compensated cirrhosis: UNITY-2 Phase 3 SVR12. In: 65th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2014), Boston, MA, November 7–12, 2014. Abstract LB-2.Google Scholar]. The sample size is also insufficient for detection of low-frequency safety-related events; however, this limitation is mitigated to some extent by the similarly favorable safety profile observed with this regimen in larger populations of patients with genotype 1 infection [7Everson GT, Sims KD, Thuluvath PJ, Lawitz E, Hassanein T, Rodriguez-Torrez M et al. Phase 2b study of the interferon-free and ribavirin-free combination of daclatasvir, asunaprevir, and BMS-791325 for 12 weeks in treatment-naive patients with chronic HCV genotype 1 infection. In: 64th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2013) Washington, DC, November 1–5, 2013. Abstract LB-1.Google Scholar, 11Muir A, Poordad F, Lalezari JP, Everson GT, Dore GJ, Kwo P et al. All-oral fixed-dose combination therapy with daclatasvir/asunaprevir/BMS-791325, +/− ribavirin, for patients with chronic HCV genotype 1 infection and compensated cirrhosis: UNITY-2 Phase 3 SVR12. In: 65th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2014), Boston, MA, November 7–12, 2014. Abstract LB-2.Google Scholar, 12Poordad F, Sievert W, Mollison L, Brau N, Levin J, Sepe T et al. All-oral fixed-dose combination therapy with daclatasvir/asunaprevir/BMS-791325 for non-cirrhotic patients with HCV genotype 1 infection: UNITY-1 Phase 3 SVR12 results. 65th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD 2014), Boston, MA, November 7–12, 2014. Abstract LB-7.Google Scholar]. In summary, in this exploratory evaluation, 12 weeks of treatment with the IFN- and ribavirin-free combination of daclatasvir, asunaprevir and beclabuvir was well tolerated and resulted in a high rate of SVR in previously untreated patients with chronic HCV genotype 4 infection, consistent with previous results in genotype 1. Conflict of interestT. Hassanein has received consulting fees from Bristol-Myers Squibb. E. Lawitz has received research grants from AbbVie, Achillion, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Idenix, Janssen, Merck, Novartis, Presidio, Roche, Santaris, and Vertex, and consulting fees and/or non-financial support from AbbVie, Achillion, BioCryst, Biotica, Enanta, Gilead Sciences, Idenix, Janssen, Kadmon, Merck, Novartis, Presidio, Santaris, Theravance, and Vertex. L. Webster has received consulting fees from AcelRx, Acura, AstraZeneca, BioDelivery Sciences International, Boehringer Ingelheim, Boston Scientific, and Collegium. Z. Younossi has received consulting fees from Salix, Janssen, Vertex, Gilead Sciences, Enterome, and Coneatus. P.J. Thuluvath has received research grant support from Bristol-Myers Squibb, Gilead Sciences, Vertex, Isai, AbbVie, and Salix, and speaker fees from Gilead Sciences, Onyx, AbbVie, and Bayer. K.D. Sims, B. Rege, N. Zhou, M. Wind-Rotolo, E. Chung, and D.M. Grasela are employees and shareholders of Bristol-Myers Squibb. H. Zhou, F. McPhee, A. Griffies, and D.F. Gardiner are employees of Bristol-Myers Squibb. M. Bennett, N. Gitlin, T. Nguyen, and H. Schwartz have no conflicts of interest to disclose. T. Hassanein has received consulting fees from Bristol-Myers Squibb. E. Lawitz has received research grants from AbbVie, Achillion, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Idenix, Janssen, Merck, Novartis, Presidio, Roche, Santaris, and Vertex, and consulting fees and/or non-financial support from AbbVie, Achillion, BioCryst, Biotica, Enanta, Gilead Sciences, Idenix, Janssen, Kadmon, Merck, Novartis, Presidio, Santaris, Theravance, and Vertex. L. Webster has received consulting fees from AcelRx, Acura, AstraZeneca, BioDelivery Sciences International, Boehringer Ingelheim, Boston Scientific, and Collegium. Z. Younossi has received consulting fees from Salix, Janssen, Vertex, Gilead Sciences, Enterome, and Coneatus. P.J. Thuluvath has received research grant support from Bristol-Myers Squibb, Gilead Sciences, Vertex, Isai, AbbVie, and Salix, and speaker fees from Gilead Sciences, Onyx, AbbVie, and Bayer. K.D. Sims, B. Rege, N. Zhou, M. Wind-Rotolo, E. Chung, and D.M. Grasela are employees and shareholders of Bristol-Myers Squibb. H. Zhou, F. McPhee, A. Griffies, and D.F. Gardiner are employees of Bristol-Myers Squibb. M. Bennett, N. Gitlin, T. Nguyen, and H. Schwartz have no conflicts of interest to disclose. Financial supportThis study was entirely funded by the sponsor, Bristol-Myers Squibb. Editorial assistance with the manuscript was provided by Richard Boehme, PhD, and Nick Fitch, PhD, of Articulate Science, with funding from the sponsor. This study was entirely funded by the sponsor, Bristol-Myers Squibb. Editorial assistance with the manuscript was provided by Richard Boehme, PhD, and Nick Fitch, PhD, of Articulate Science, with funding from the sponsor. Authors' contributionsK.D. Sims, D.M. Grasela, E. Chung, A. Griffies, and D.F. Gardiner designed the study. T. Hassanein, M. Bennett, N. Gitlin, E. Lawitz, T. Nguyen, L. Webster, Z. Younossi, H. Schwartz, and P.J. Thuluvath recruited patients and obtained data. H. Zhou, B. Rege, F. McPhee, N. Zhou, and M. Wind-Rotolo analyzed the data. All authors interpreted the data, participated in writing the manuscript, and approved the final version of the manuscript. K.D. Sims, D.M. Grasela, E. Chung, A. Griffies, and D.F. Gardiner designed the study. T. Hassanein, M. Bennett, N. Gitlin, E. Lawitz, T. Nguyen, L. Webster, Z. Younossi, H. Schwartz, and P.J. Thuluvath recruited patients and obtained data. H. Zhou, B. Rege, F. McPhee, N. Zhou, and M. Wind-Rotolo analyzed the data. All authors interpreted the data, participated in writing the manuscript, and approved the final version of the manuscript. Supplementary data Download .pdf (.12 MB) Help with pdf files Supplementary Fig. 1 and Tables 1 and 2 Download .pdf (.12 MB) Help with pdf files Supplementary Fig. 1 and Tables 1 and 2