Andrew Dellinger

Relationship Between Methylome and Transcriptome in Patients With Nonalcoholic Fatty Liver Disease

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.

Hepatic Gene Expression Profiles Differentiate Presymptomatic Patients With Mild Versus Severe Nonalcoholic Fatty Liver Disease

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)

Gene Expression Profile in Human Trabecular Meshwork From Patients With Primary Open-Angle Glaucoma

PURPOSE To identify the specific genes in human trabecular meshwork (TM) related to POAG. METHODS Primary open-angle glaucoma TM specimens were obtained from routine trabeculectomy surgery. Nonglaucomatous control TM specimens were dissected from donor eyes using the same approach as a standard trabeculectomy. All cases were screened for myocilin (MYOC) mutations. Total RNA was extracted, labeled, and hybridized to Illumina HumanWG-6 BeadChips. Expression data were normalized and analyzed using the R package limma in Bioconductor. Pathway analyses were performed using DAVID Bioinformatics Resources. RESULTS Our study included surgical TM specimens from 15 cases and 13 controls. One case was identified with a heterozygous Q368X MYOC mutation. If TMs were available from both eyes in an individual, the expression data were combined for analysis. The following three comparisons were performed for differential analyses: (1) MYOC POAG case versus 14 non-MYOC POAG cases, (2) MYOC POAG case versus 13 controls, and (3) 14 non-MYOC POAG cases versus 13 controls. Limited by one MYOC case in comparisons 1 and 2, expression changes were reported comparing the fold changes but without P values. Comparison 3 identified 483 genes, including 36 components of TM exosomes. Gene ontology analysis identified several enriched functional clusters, including cell adhesion, extracellular matrix, and secretion. CONCLUSIONS This is the largest TM expression study of POAG cases and controls performed to date and represents the first report of TM expression in a patient having POAG with a Q368X MYOC mutation. Our data suggest the potential role of endocytic and exosome pathways in the pathogenesis of POAG.

A phase I study of ABT-510 plus bevacizumab in advanced solid tumors.

Targeting multiple regulators of tumor angiogenesis have the potential to improve treatment efficacy. Bevacizumab is a monoclonal antibody directed against vascular endothelial growth factor and ABT‐510 is a synthetic analog of thrombospondin, an endogenous angiogenesis inhibitor. Dual inhibition may result in additional benefit. We evaluated the safety, tolerability, and efficacy of the combination of bevacizumab plus ABT‐510 in patients with refractory solid tumors. We also explored the effects of these agents on plasma‐based biomarkers and wound angiogenesis. Thirty‐four evaluable subjects were enrolled and received study drug. Therapy was well tolerated; minimal treatment‐related grade 3/4 toxicity was observed. One patient treated at dose level 1 had a partial response and five other patients treated at the recommended phase II dose had prolonged stable disease for more than 1 year. Biomarker evaluation revealed increased levels of D‐dimer, von Willebrand factor, placental growth factor, and stromal‐derived factor 1 in response to treatment with the combination of bevacizumab and ABT‐510. Data suggest that continued evaluation of combination antiangiogenesis therapies may be clinically useful.

Modulation of circulating protein biomarkers following TRC105 (anti-endoglin antibody) treatment in patients with advanced cancer

TRC105 is an endoglin‐targeting drug that possesses anti‐angiogenic and antitumor potential. Analysis of the initial phase I trial of TRC105 demonstrated good tolerability and efficacy in cancer patients. In this report, we analyzed multiple circulating biomarkers at baseline, cycle 2 day 1 (C2D1), and end of study (EOS) for each patient. The baseline level and the fold change from baseline to both C2D1 and EOS for each marker were statistically analyzed. At C2D1, seven markers were significantly downregulated (angiopoietin‐2 [Ang‐2], insulin‐like growth factor‐binding protein‐3 [IGFBP‐3], plasminogen activator inhibitor‐1 [PAI‐1] total, platelet‐derived growth factor [PDGF]‐AA, PDGF‐BB, thrombospondin‐1 [TSP‐1], and vascular endothelial growth factor [VEGF]‐D). Meanwhile, seven markers were upregulated by C2D1 (E‐Cadherin, soluble Endoglin [sEnd], E‐Selectin, interleukin‐6 [IL‐6], osteopontin [OPN], TSP‐2, and von Willebrand factor [vWF]). At EOS, seven markers were upregulated including Ang‐2, C‐reactive protein (CRP), intercellular adhesion molecule‐1 (ICAM‐1), IGFBP‐1, IL‐6, TSP‐2, and vascular cell adhesion molecule‐1 (VCAM‐1). A statistical trend was also seen for increases of VEGF‐A and placenta growth factor (PlGF) at EOS. Throughout treatment, sEnd levels significantly increased, an observation that was recapitulated in cultured endothelial cells. This is the first report of plasma‐based biomarkers in patients receiving TRC105. TRC105 treatment by C2D1 was associated with decreases in several angiogenic factors, including Ang‐2, PDGF isoforms, and VEGF isoforms, offering insight into the mechanisms underlying TRC105s anti‐angiogenic, antitumor function. Increases in sEnd were the most significant of all observed biomarker changes and may reflect direct drug effects. Additionally, biomarker changes in response to TRC105 are distinct from those seen in patients treated with VEGF‐targeting drugs, suggesting the possible utility of combining these two classes of angiogenesis inhibitors in patients.

Transcriptome profiling of genes involved in neural tube closure during human embryonic development using long serial analysis of gene expression (long‐SAGE)

BACKGROUND Neural tube defects (NTDs) are common human birth defects with a complex etiology. To develop a comprehensive knowledge of the genes expressed during normal neurulation, we established transcriptomes from human neural tube fragments during and after neurulation using long Serial Analysis of Gene Expression (long-SAGE). METHODS Rostral and caudal neural tubes were dissected from normal human embryos aged between 26 and 32 days of gestation. Tissues from the same region and Carnegie stage were pooled (n ≥ 4) and total RNA extracted to construct four long-SAGE libraries. Tags were mapped using the UniGene Homo sapiens 17 bp tag-to-gene best mapping set. Differentially expressed genes were identified by chi-square or Fishers exact test, and validation was performed for a subset of those transcripts using in situ hybridization. In silico analyses were performed with BinGO and EXPANDER. RESULTS We observed most genes to be similarly regulated in rostral and caudal regions, but expression profiles differed during and after closure. In silico analysis found similar enrichments in both regions for biologic process terms, transcription factor binding and miRNA target motifs. Twelve genes potentially expressing alternate isoforms by region or developmental stage, and the microRNAs miR-339-5p, miR-141/200a, miR-23ab, and miR-129/129-5p are among several potential candidates identified here for future research. CONCLUSIONS Time appears to influence gene expression in the developing central nervous system more than location. These data provide a novel complement to traditional strategies of identifying genes associated with human NTDs and offer unique insight into the genes associated with normal human neurulation.

Principal-component-based multivariate regression for genetic association studies of metabolic syndrome components.

BackgroundQuantitative traits often underlie risk for complex diseases. For example, weight and body mass index (BMI) underlie the human abdominal obesity-metabolic syndrome. Many attempts have been made to identify quantitative trait loci (QTL) over the past decade, including association studies. However, a single QTL is often capable of affecting multiple traits, a quality known as gene pleiotropy. Gene pleiotropy may therefore cause a loss of power in association studies focused only on a single trait, whether based on single or multiple markers.ResultsWe propose using principal-component-based multivariate regression (PCBMR) to test for gene pleiotropy with comprehensive evaluation. This method generates one or more independent canonical variables based on the principal components of original traits and conducts a multivariate regression to test for association with these new variables. Systematic simulation studies have shown that PCBMR has great power. PCBMR-based pleiotropic association studies of abdominal obesity-metabolic syndrome and its possible linkage to chromosomal band 3q27 identified 11 susceptibility genes with significant associations. Whereas some of these genes had been previously reported to be associated with metabolic traits, others had never been identified as metabolism-associated genes.ConclusionsPCBMR is a computationally efficient and powerful test for gene pleiotropy. Application of PCBMR to abdominal obesity-metabolic syndrome indicated the existence of gene pleiotropy affecting this syndrome.

Integrative Pathway Analysis Using Graph-Based Learning with Applications to TCGA Colon and Ovarian Data

Recent method development has included multi-dimensional genomic data algorithms because such methods have more accurately predicted clinical phenotypes related to disease. This study is the first to conduct an integrative genomic pathway-based analysis with a graph-based learning algorithm. The methodology of this analysis, graph-based semi-supervised learning, detects pathways that improve prediction of a dichotomous variable, which in this study is cancer stage. This analysis integrates genome-level gene expression, methylation, and single nucleotide polymorphism (SNP) data in serous cystadenocarcinoma (OV) and colon adenocarcinoma (COAD). The top 10 ranked predictive pathways in COAD and OV were biologically relevant to their respective cancer stages and significantly enhanced prediction accuracy and area under the ROC curve (AUC) when compared to single data-type analyses. This method is an effective way to simultaneously predict binary clinical phenotypes and discover their biological mechanisms.

Direct Evidence of Target Inhibition with anti-VEGF, EGFR, and mTOR Therapies in a Clinical Model of Wound Healing

Purpose: In early clinical testing, most novel targeted anticancer therapies have limited toxicities and limited efficacy, which complicates dose and schedule selection for these agents. Confirmation of target inhibition is critical for rational drug development; however, repeated tumor biopsies are often impractical and peripheral blood mononuclear cells and normal skin are often inadequate surrogates for tumor tissue. Based upon the similarities of tumor and wound stroma, we have developed a clinical dermal granulation tissue model to evaluate novel targeted therapies. Experimental Design: A 4-mm skin punch biopsy was used to stimulate wound healing and a repeat 5-mm punch biopsy was used to harvest the resulting granulation tissue. This assay was performed at pretreatment and on-treatment evaluating four targeted therapies, bevacizumab, everolimus, erlotinib, and panitumumab, in the context of three different clinical trials. Total and phosphorylated levels VEGFR2, S6RP, and EGFR were evaluated using ELISA-based methodologies. Results: Significant and consistent inhibition of the VEGF pathway (using VEGFR2 as the readout) was observed in granulation tissue biopsies from patients treated with bevacizumab and everolimus. In addition, significant and consistent inhibition of the mTOR pathway (using S6RP as the readout) was observed in patients treated with everolimus. Finally, significant inhibition of the EGFR pathway (using EGFR as the readout) was observed in patients treated with panitumumab, but this was not observed in patients treated with erlotinib. Conclusions: Molecular analyses of dermal granulation tissue can be used as a convenient and quantitative pharmacodynamic biomarker platform for multiple classes of targeted therapies. Clin Cancer Res; 21(15); 3442–52. ©2015 AACR.

Coffee's Beneficial Effect on Liver Disease Confirmed in NASH Cohort, but Only Partially Confirmation of In Vitro Pre-Described Differentially Expressed Genes in This Patient Cohort

Background and Aim: Recent studies have implicated several important hepatic cellular processes and signaling pathways that are affected by abnormal lipid metabolism, resulting in specific biochemical, histological, and clinical changes associated with NAFLD. We established highly sensitive and specific analysis of serum biomarkers for comprehensive view of the change in cholesterol and fatty acid metabolism by HPLC-ESI-MS/MS. By using this method, we monitored lipid metabolism in NAFLD patients before and during administration of pitavastatin. Materials and Methods: Serum samples were obtained from patients with NAFLD enrolled in multicenter case control study (n=15) at baseline, 3 months (mo), and 12 mo from initiation of pitavastatin treatment(1~2mg/day). Sex and age matched control (CTL) samples were obtained from 36 healthy volunteers with none of obesity, hyperlipidemia, and liver dysfunction. Internal standards were added to 10μL of serum. After extraction and derivatization, the samples were analyzed by LC-MS/MS equipped with reversed C18 column. Results: Markers of intestinal cholesterol absorption, serum sitosterol (CTL vs. NAFLD: 4.04±1.79 vs 1.69±0.68 ng/mL, P<0.001) and campesterol (4.77±2.13 vs. 1.71±0.79 ng/mL, P<0.001) were significantly lower in patients with NAFLD compared to CTL. Markers of LXR activity, serum 24S-hydroxy cholesterol (69.53±19.8 vs. 92.29±30.5 ng/mL, P<0.05), and 27-hydroxy cholesterol (136.0±41.94 vs. 244.7± 59.34 ng/mL) were significantly higher in patients with NAFLD compared to CTL. Lathosterol, a marker of cholesterol synthesis, tended to be higher in NAFLD but not significantly different. By pitavastatin administration, decrease of cholesterol synthesis was found during observation period. Decrease of LXR was not found at 3 mo but 12 mo follow up. Alterations in bile acid synthesis, cholesterol absorption and fatty acid synthesis were not significant over observation period of time. On the other hands, analysis of these markers in a subgroup with (responder) and a subgroup without (non-responder) decreasing ALT level revealed that decrease of bile acid synthesis (responder vs. non-responder: -22.3 vs. 69.0% of pre-treatment level) and DNL (-7.47 vs. 31.6%) were prominent in responder group, while there was no significant change in decrease of cholesterol synthesis in both group (-60.3% vs. -70.7%). Conclusions: In patients with NAFLD, intestinal absorption was inhibited by the activation of LXR. Treatment of pitavastatin lowered cholesterol synthesis and LXR activity in all patients, however, suppression of bile acid as well as DNL were observed only in patients with improved biochemical data. More research work is needed to understand the clinical meaning of these findings, which may help to understand the role of lipid lowering agents in the treatment of NAFLD.