Background & Aims Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. Methods We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0−1 (mild) and F3−4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. Results Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed. Conclusions Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Background: Propensity score (PS) analysis is increasingly being used in observational studies, especially in some cancer studies where random assignment is not feasible. This systematic review evaluates the use and reporting quality of PS analysis in oncology studies. Methods: We searched PubMed to identify the use of PS methods in cancer studies (CS) and cancer surgical studies (CSS) in major medical, cancer, and surgical journals over time and critically evaluated 33 CS published in top medical and cancer journals in 2014 and 2015 and 306 CSS published up to November 26, 2015, without earlier date limits. The quality of reporting in PS analysis was evaluated. It was also compared over time and among journals with differing impact factors. All statistical tests were two-sided. Results: More than 50% of the publications with PS analysis from the past decade occurred within the past two years. Of the studies critically evaluated, a considerable proportion did not clearly provide the variables used to estimate PS (CS 12.1%, CSS 8.8%), incorrectly included non baseline variables (CS 3.4%, CSS 9.3%), neglected the comparison of baseline characteristics (CS 21.9%, CSS 15.6%), or did not report the matching algorithm utilized (CS 19.0%, CSS 36.1%). In CSS, the reporting of the matching algorithm improved in 2014 and 2015 (P = .04), and the reporting of variables used to estimate PS was better in top surgery journals (P = .008). However, there were no statistically significant differences for the inclusion of non baseline variables and reporting of comparability of baseline characteristics. Conclusions: The use of PS in cancer studies has dramatically increased recently, but there is substantial room for improvement in the quality of reporting even in top journals. Herein we have proposed reporting guidelines for PS analyses that are broadly applicable to different areas of medical research that will allow better evaluation and comparison across studies applying this approach.

Estrogens inhibit stellate cell activation and fibrogenesis. Thus, gender and reproductive states may influence the degree of fibrosis in patients with nonalcoholic steatohepatitis (NASH). To investigate the association between gender, menopause, and the severity of liver fibrosis in patients with NASH, we analyzed 541 adult patients enrolled from our Duke Liver Clinics (n = 338) and the Duke Metabolic and Weight Loss Surgery Program (n = 203) who had a histologic diagnosis of NASH. Multiple ordinal logistic regression models were used to assess the association between gender, menopause, and severity of liver fibrosis. Overall, men, premenopausal, and postmenopausal women composed 35.1%, 28.4%, and 36.5% of the population, respectively. The mean age was 48 years and 22% had advanced fibrosis. After adjusting for covariates (enrolling site, grades of portal inflammation, and hepatocyte ballooning) and potential confounders (race, body mass index, diabetes/prediabetes, hypertension), adjusted cumulative odd ratio (ACOR) and 95% confidence interval (CI) for greater fibrosis severity was 1.4 (0.9, 2.1) (P = 0.17) for postmenopausal women and 1.6 (1.0, 2.5) (P = 0.03) for men, having premenopausal women as a reference. There was borderline interaction between gender and age group divided by age 50, the average age at menopause in the U.S. (P = 0.08): ACOR and 95% CI of having greater fibrosis severity in men compared to women was 1.8 (1.1, 2.9) for patients with age <50 years (P = 0.02) and 1.2 (0.7, 2.1) for patients with age ≥50 years (P = 0.59). Conclusion: Men are at a higher risk of having more severe fibrosis compared to women before menopause, while postmenopausal women have a similar severity of liver fibrosis compared to men. These findings may be explained by the protective effects of estrogen against fibrogenesis. (HEPATOLOGY 2014;59:1406-1414)

Clinicians rely upon the severity of liver fibrosis to segregate patients with well-compensated nonalcoholic fatty liver disease (NAFLD) into subpopulations at high- versus low-risk for eventual liver-related morbidity and mortality. We compared hepatic gene expression profiles in high- and low-risk NAFLD patients to identify processes that distinguish the two groups and hence might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low-risk, “mild” NAFLD patients (fibrosis stage 0–1; n = 40) and high-risk, “severe” NAFLD patients (fibrosis stage 3–4; n = 32). Findings were validated in a second, independent cohort and confirmed by real-time polymerase chain reaction and immunohistochemistry (IHC). As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64-gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20-gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism-related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and IHC confirmed deregulation of metabolic and regenerative pathways in severe NAFLD and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer. Conclusion : By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD-related morbidity and mortality. (Hepatology 2014;59:471–482)

Objective Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. Design The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. Results When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18‒/‒ and CD1d‒/‒ mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. Conclusion Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.

The gold standard endpoint in clinical trials of chemotherapy and radiotherapy for lung cancer is overall survival. Although reliable and simple to measure, this endpoint takes years to observe. Surrogate endpoints that would enable earlier assessments of treatment effects would be useful. We assessed the correlations between potential surrogate endpoints and overall survival at individual and trial levels. We analysed individual patients' data from 15,071 patients involved in 60 randomised clinical trials that were assessed in six meta-analyses. Two meta-analyses were of adjuvant chemotherapy in non-small-cell lung cancer, three were of sequential or concurrent chemotherapy, and one was of modified radiotherapy in locally advanced lung cancer. We investigated disease-free survival (DFS) or progression-free survival (PFS), defined as the time from randomisation to local or distant relapse or death, and locoregional control, defined as the time to the first local event, as potential surrogate endpoints. At the individual level we calculated the squared correlations between distributions of these three endpoints and overall survival, and at the trial level we calculated the squared correlation between treatment effects for endpoints. In trials of adjuvant chemotherapy, correlations between DFS and overall survival were very good at the individual level (ρ(2)=0.83, 95% CI 0.83-0.83 in trials without radiotherapy, and 0.87, 0.87-0.87 in trials with radiotherapy) and excellent at trial level (R(2)=0.92, 95% CI 0.88-0.95 in trials without radiotherapy and 0.99, 0.98-1.00 in trials with radiotherapy). In studies of locally advanced disease, correlations between PFS and overall survival were very good at the individual level (ρ(2) range 0.77-0.85, dependent on the regimen being assessed) and trial level (R(2) range 0.89-0.97). In studies with data on locoregional control, individual-level correlations were good (ρ(2)=0.71, 95% CI 0.71-0.71 for concurrent chemotherapy and ρ(2)=0.61, 0.61-0.61 for modified vs standard radiotherapy) and trial-level correlations very good (R(2)=0.85, 95% CI 0.77-0.92 for concurrent chemotherapy and R(2)=0.95, 0.91-0.98 for modified vs standard radiotherapy). We found a high level of evidence that DFS is a valid surrogate endpoint for overall survival in studies of adjuvant chemotherapy involving patients with non-small-cell lung cancers, and PFS in those of chemotherapy and radiotherapy for patients with locally advanced lung cancers. Extrapolation to targeted agents, however, is not automatically warranted. Programme Hospitalier de Recherche Clinique, Ligue Nationale Contre le Cancer, British Medical Research Council, Sanofi-Aventis.