Megan Comerford

Relationship Between Changes in Serum Levels of Keratin 18 and Changes in Liver Histology in Children and Adults With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Cross-sectional studies have associated serum levels of the keratin 18 (K18) fragment with histologic features of liver in individuals with nonalcoholic fatty liver disease (NAFLD). We investigated the relationship between changes in serum levels of K18 and changes in liver histology in adults and children with NAFLD. METHODS We measured levels of K18 in stored serum samples collected at baseline and various time points from 231 adults with nonalcoholic steatohepatitis and 152 children with NAFLD who participated in 2 separate prospective randomized clinical trials. Liver biopsy specimens collected at baseline and week 96 were reviewed centrally. RESULTS There were greater decreases in serum levels of K18 in adults with histologic improvement at week 96 than in those without histologic improvement at week 16 (decrease, 193 ± 293 vs 139 ± 467 U/L; P < .001), week 48 (decrease, 232 ± 360 vs 113 ± 425 U/L; P < .001), or week 96 (decrease, 269 ± 368 vs 97 ± 400 U/L; P < .001). There were greater decreases in serum levels of K18 in children with histologic improvements than in those without histologic improvements at week 48 (decrease, 197 ± 467 vs 47 ± 350 U/L; P = .005) and week 96 (decrease, 206 ± 432 vs 2 ± 474 U/L; P < .001). However, reductions in serum levels of K18 were not better than reductions in levels of alanine aminotransferase in identifying adults with histologic improvement (area under the receiver operator characteristic [AUROC], 0.71; 95% confidence interval [CI], 0.63-0.80; vs AUROC, 0.68; 95% CI, 0.61-0.79; P = .34) or children (AUROC, 0.72; 95% CI, 0.63-0.81; vs AUROC, 0.79; 95% CI, 0.70-0.87; P = .42). CONCLUSIONS Decreases in serum levels of K18 are associated strongly with improved liver histologies in adults or children with NAFLD. However, reductions in K18 do not perform better than those in alanine aminotransferase level in identifying histologic changes in NAFLD.

The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitis

Intestinal dysbiosis is implicated in alcoholic hepatitis (AH). However, changes in the circulating microbiome, its association with the presence and severity of AH, and its functional relevance in AH is unknown. Qualitative and quantitative assessment of changes in the circulating microbiome were performed by sequencing bacterial DNA in subjects with moderate AH (MAH) (n = 18) or severe AH (SAH) (n = 19). These data were compared with heavy drinking controls (HDCs) without obvious liver disease (n = 19) and non–alcohol‐consuming controls (NACs, n = 20). The data were related to endotoxin levels and markers of monocyte activation. Linear discriminant analysis effect size (LEfSe) analysis, inferred metagenomics, and predictive functional analysis using PICRUSt were performed. There was a significant increase in 16S copies/ng DNA both in MAH (P < 0.01) and SAH (P < 0.001) subjects. Compared with NACs, the relative abundance of phylum Bacteroidetes was significantly decreased in HDCs, MAH, and SAH (P < 0.001). In contrast, all alcohol‐consuming groups had enrichment with Fusobacteria; this was greatest for HDCs and decreased progressively in MAH and SAH. Subjects with SAH had significantly higher endotoxemia (P = 0.01). Compared with alcohol‐consuming groups, predictive functional metagenomics indicated an enrichment of bacteria with genes related to methanogenesis and denitrification. Furthermore, both HDCs and SAH showed activation of a type III secretion system that has been linked to gram‐negative bacterial virulence. Metagenomics in SAH versus NACs predicted increased isoprenoid synthesis via mevalonate and anthranilate degradation, known modulators of gram‐positive bacterial growth and biofilm production, respectively. Conclusion: Heavy alcohol consumption appears to be the primary driver of changes in the circulating microbiome associated with a shift in its inferred metabolic functions. (Hepatology 2018;67:1284‐1302).

Investigation Gone Viral: Application of the Social Mediasphere in Research

The research paradigm in the United States remains burdened by numerous obstacles impeding the progress of scientific investigation.1 Barriers to effective and efficient conduct of academic research include growing costs, delayed results, adequate staffing, and regulatory encumbrances.2–5 Beyond these system constraints, patient recruitment in research studies can be time-intensive, costly, and limited by minimal participant diversity.6,7 The social mediasphere, an intertwining universe of online social media applications, may represent a new model in research methodology that will bridge current research challenges in all medical fields. Investigators and study staff will require a fundamental appreciation of social media structure, existing methodology, and advantages and limitations in order to effectively conduct research with this novel strategy.

518 Changes in Serum Cytokeratin 18 Levels Significantly Predict Changes in Liver Histology in Adults With Nonalcoholic Steatohepatitis: Results From the Pivens Trial

Background: Previous cross-sectional studies have shown that serum levels of cytokeratin fragments (CK18) are associated with the presence of steatohepatitis in individuals with nonalcoholic fatty liver disease (NAFLD). However, it is unknown if serial serum CK18 levels can predict longitudinal changes in liver histology in NAFLD. Aim: To determine the degree to which changes in serum CK18 associate with changes in liver histology over a 96-week period in adult patients with nonalcoholic steatohepatitis (NASH). Methods: Serum CK18 levels were measured at baseline and 16, 48, and 96 weeks in 231 of the 247 adults with NASH who participated in the PIVENS trial which investigated the efficacy of pioglitazone vs. vitamin E vs. placebo in non-diabetic individuals with histologically confirmed NASH over a 96-week period. Liver biopsies at baseline and after 96 weeks of treatment were centrally evaluated by the NASH CRN Pathology Committee. Multiple logistic (for categorical outcomes) and linear (for continuous outcome variables) regression models were used to determine the association between changes in histological features, ALT, and % collagen assessed by Sirius red staining, and change in CK18 levels, controlling for baseline CK18 levels and treatment group. Results: At baseline, CK18 levels among the 3 treatment groups were similar (placebo: 440± 350 U/L, vit E: 510± 350 U/L, and pioglitazone: 490± 410 U/L). Compared to placebo, serum CK18 levels among individuals treated with vit E were reduced at week 16 (mean change from baseline -160± 300 vs. -50 ±380 U/L, p= 0.02), week 48 (-220± 390 vs. -10± 370 U/L, p=0.009), and week 96 (-200± 400 vs. 30± 400 U/L, p=0.009). Similarly, compared to placebo, serum CK18 levels among individuals treated with pioglitazone were reduced at week 16 (mean change from baseline -240 ± 390 vs. -50± 380 U/L, p,=0.001), week 48 (-180± 370 vs. -10± 370 U/L, p=0.001, and week 96 (-260 ±400 vs. 30 ± 400 U/L, p=0.001). Strong correlations were seen with change in CK18 levels and the primary histologic endpoint, resolution of NASH, and individual histologic features of NASH (Table 1). In addition, change in CK18 levels strongly correlated with change in ALT levels (b=0.06, 95% CI: 0.04, 0.08; P,0.001).Percent collagen measurements were not related to change in CK18 (b=-0.17 %/100*U/L , 95% CI:-0.70, 0.37; P= 0.54). Summary: (1) Compared to placebo, treatment with vit E or pioglitazone had significant reduction in serum CK18 levels. (2) Changes in serum CK18 correlated with histologic improvement in non-diabetic adults with NASH treated with vitamin E or pioglitazone. Conclusion: Serum CK18 is a potential useful surrogate marker for detection of improvement in clinical trials.

Perilipin Staining Distinguishes Between Steatosis and Nonalcoholic Steatohepatitis in Adults and Children

*Perelman School of Medicine, Department of Medicine, Gastroenterology and Hepatology Division, Division of Endocrinology, Diabetes and Metabolism, University of Pennsylvania, Philadelphia, Pennsylvania; School of Medical Sciences, University of Hyderabad, Hyderabad, India; kDivision of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, Indiana; Division of Endocrinology, Diabetes and Metabolism, Johns Hopkins University School of Medicine, Baltimore, Maryland

Risk factors for hepatocellular carcinoma in cirrhosis due to nonalcoholic fatty liver disease: A multicenter, case-control study

AIM To identify risk factors associated with hepatocellular carcinoma (HCC), describe tumor characteristics and treatments pursed for a cohort of individuals with nonalcoholic steatohepatitis (NASH) cirrhosis. METHODS We conducted a retrospective case-control study of a well-characterized cohort of patients among five liver transplant centers with NASH cirrhosis with (cases) and without HCC (controls). RESULTS Ninety-four cases and 150 controls were included. Cases were significantly more likely to be male than controls (67% vs 45%, P < 0.001) and of older age (61.9 years vs 58 years, P = 0.002). In addition, cases were more likely to have had complications of end stage liver disease (83% vs 71%, P = 0.032). On multivariate analysis, the strongest association with the presence of HCC were male gender (OR 4.3, 95%CI: 1.83-10.3, P = 0.001) and age (OR = 1.082, 95%CI: 1.03-1.13, P = 0.001). Hispanic ethnicity was associated with a decreased prevalence of HCC (OR = 0.3, 95%CI: 0.09-0.994, P = 0.048). HCC was predominantly in the form of a single lesion with regional lymph node(s) and distant metastasis in only 2.6% and 6.3%, respectively. Fifty-nine point three percent of individuals with HCC underwent locoregional therapy and 61.5% underwent liver transplantation for HCC. CONCLUSION Male gender, increased age and non-Hispanic ethnicity are associated with HCC in NASH cirrhosis. NASH cirrhosis associated HCC in this cohort was characterized by early stage disease at diagnosis and treatment with locoregional therapy and transplant.

Leveraging Social Networking Sites for an Autoimmune Hepatitis Genetic Repository: Pilot Study to Evaluate Feasibility

Background Conventional approaches to participant recruitment are often inadequate in rare disease investigation. Social networking sites such as Facebook may provide a vehicle to circumvent common research limitations and pitfalls. We report our preliminary experience with Facebook-based methodology for participant recruitment and participation into an ongoing study of autoimmune hepatitis (AIH). Objective The goal of our research was to conduct a pilot study to assess whether a Facebook-based methodology is capable of recruiting geographically widespread participants into AIH patient-oriented research and obtaining quality phenotypic data. Methods We established a Facebook community, the Autoimmune Hepatitis Research Network (AHRN), in 2014 to provide a secure and reputable distillation of current literature and AIH research opportunities. Quarterly advertisements for our ongoing observational AIH study were posted on the AHRN over 2 years. Interested and self-reported AIH participants were subsequently enrolled after review of study materials and completion of an informed consent by our study coordinator. Participants returned completed study materials, including epidemiologic questionnaires and genetic material, to our facility via mail. Outside medical records were obtained and reviewed by a study physician. Results We successfully obtained all study materials from 29 participants with self-reported AIH within 2 years from 20 different states. Liver biopsy results were available for 90% (26/29) of participants, of which 81% (21/29) had findings consistent with AIH, 15% (4/29) were suggestive of AIH with features of primary biliary cholangitis (PBC), and 4% (1/29) had PBC alone. A total of 83% (24/29) had at least 2 of 3 proposed criteria: positive autoimmune markers, consistent histologic findings of AIH on liver biopsy, and reported treatment with immunosuppressant medications. Self-reported and physician records were discrepant for immunosuppressant medications or for AIH/PBC diagnoses in 4 patients. Conclusions Facebook can be an effective ancillary tool for facilitating patient-oriented research in rare diseases. A social media-based approach transcends established limitations in rare disease research and can further develop research communities.

Extrahepatic Autoimmune Diseases are Prevalent in Autoimmune Hepatitis Patients and their First-Degree Relatives (Preprint)

Background Concurrent autoimmune illnesses contribute to increased medical burden and reduced quality of life in patients with autoimmune hepatitis (AIH). The frequency of coexisting autoimmune conditions among North American patients with AIH and their families remains incomplete. Challenges associated with disease capture in the electronic medical record, high study costs, and geographic spread of patients are formidable barriers to understanding the extent of concurrent autoimmune conditions in these groups. Objective This objective of this study was to examine the frequency of extrahepatic autoimmune diseases (EHAD) among AIH cases and healthy controls as well as their first-degree relatives using social networking sites (SNS). Methods We developed a 53-question survey detailing the history of autoimmune diseases. A survey link was posted at routine intervals within specific Web-based cohorts on SNS. Healthy controls, without self-reported autoimmune liver disease, were recruited from Amazon’s Mechanical Turk. Continuous variables were summarized using medians and P values obtained with the Wilcoxon rank-sum test. Categorical variables were compared using the chi-square test. Results Compared with controls (n=1162), cases (n=306) were more likely to be older (median age: 49 vs 33 years), female (284/306, 92.81% vs 955/1162, 82.18%), and have an EHAD (128/306, 41.83% vs 218/1162, 18.76%; P=.001). The most frequent EHADs among cases were thyroid disease (49/306, 16.01% ), Sjögren syndrome (27/306, 8.82%), Raynaud phenomenon (23/306, 7.52%), and psoriasis (22/306, 7.19%). Overall, 55.88% (171/306) of cases and 35.71% (1601/4484) of controls reported at least 1 first-degree relative (FDR) with a history of EHAD (P=.001). Cases had a significantly higher risk of EHAD than controls after the adjustment for age, sex, race, and body mass index: odds ratio 2.46 (95% CI 1.8-3.3); P=.001. Conclusions Patients with AIH report higher prevalence of coexistent EHAD than healthy controls, and their FDRs are also more likely to have autoimmune disorders.

Serum high mobility group box 1 protein levels are not associated with either histological severity or treatment response in children and adults with nonalcoholic fatty liver disease

Aim Serum high mobility group box 1 protein (HMGB1) is a proinflammatory molecule that could potentially serve as a biomarker for non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) due to its correlation with degree of liver fibrosis. The aim of the current study was to examine the cross-sectional and longitudinal relationships between serum HMGB1 levels and liver histology in adults and children with NAFLD participating in two large randomized controlled trials. Methods Serum HMGB1 levels were measured at various time points in adults and children with NAFLD, who participated in PIVENS and TONIC clinical trials respectively. PIVENS trial compared vitamin E or pioglitazone to placebo in adults whereas TONIC trial compared vitamin E or metformin to placebo in children. Participants had liver biopsies at baseline and the end of treatment (96 weeks), and liver histology was reviewed by a central committee of study pathologists. Results In the cross-sectional analyses (n = 205 for PIVENS and 109 for TONIC), there was no significant relationship between serum HMGB1 levels and histological features such as steatosis, ballooning, inflammation, fibrosis, or presence of steatohepatitis in either adults or children. Serum HMGB1 levels did not change significantly during treatment either with placebo, vitamin E therapy (P = 0.81) or pioglitazone (P = 0.09) in the PIVENS trial. Similarly, serum HMGB1 levels did not change significantly during treatment either with placebo, metformin (P = 0.15) or vitamin E (P = 0.23) in the TONIC trial. In the longitudinal analyses (n = 105 for PIVENS and 109 for TONIC), changes in serum HMGB1 levels did not correlate with histologic improvement or resolution of NASH in either adults or children. There was no relationship between serum HMGB1 and ALT levels in either adults or children with NAFLD. Conclusion Serum HMGB1 levels were not associated with histological severity or treatment response in either children or adults with NAFLD.

Acute Alcoholic Hepatitis: Natural History and Predictors of Mortality Using a Multicenter Prospective Study

Objective To examine the natural history of acute alcoholic hepatitis (AH) and identify predictors of mortality for AH using data from a prospective multicenter observational study.Objective To examine the natural history of acute alcoholic hepatitis (AH) and identify predictors of mortality for AH using data from a prospective multicenter observational study. Participants and Methods We analyzed data from 164 patients with AH and 131 heavy-drinking controls with no liver disease. Participants underwent clinical/laboratory assessment at baseline and 6 and 12 months after enrollment. Multivariable analyses were conducted to identify variables associated with mortality and examine the association between coffee drinking and risk of AH. Results Thirty-six patients with AH died during follow-up, with estimated 30-day, 90-day, 180-day, and 1-year survival of 0.91 (95% CI, 0.87-0.96), 0.85 (95% CI, 0.80-0.91), 0.80 (95% CI, 0.74-0.87), and 0.75 (95% CI, 0.68-0.83), respectively. In the multivariable analysis, higher serum bilirubin level (hazard ratio [HR]=1.059; 95% CI, 1.022-1.089), lower hemoglobin level (HR=1.263; 95% CI, 1.012-1.575), and lower platelet count (HR=1.006; 95% CI, 1.001-1.012) were independently associated with mortality in AH. Compared with controls, fewer patients with AH regularly consumed coffee (20% vs 44%; P<.001), and this association between regular coffee drinking and lower risk of AH persisted after controlling for relevant covariates (odds ratio=0.26; 95% CI, 0.15-0.46). Time-dependent receiver operating characteristic curve analysis revealed that Model for End-Stage Liver Disease; Maddrey Discriminant Function; age, serum bilirubin, international normalized ratio, and serum creatinine; and Child-Pugh scores all provided similar discrimination performance at 30 days (area under the curve=0.73-0.77). Conclusion Alcoholic hepatitis remains highly fatal, with 1-year mortality of 25%. Regular coffee consumption was associated with lower risk of AH in heavy drinkers.