Cancer of the ampulla of Vater: analysis of the whole genome sequence exposes a potential therapeutic vulnerability

BACKGROUND: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers. METHODS: We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations. RESULTS: We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition. CONCLUSIONS: Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers

Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts

<p>Abstract</p> <p>Background</p> <p>Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The <it>Escherichia coli </it>S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to <it>E. coli</it>, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize <it>in vitro </it>translation for protein microarray assembly.</p> <p>Results</p> <p>Self-assembling autofluorescent protein microarrays were produced by <it>in situ </it>transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when <it>in vitro </it>translation was performed with <it>E. coli </it>S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid <it>in vitro </it>translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested.</p> <p>Conclusion</p> <p>The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the <it>in vitro </it>system by providing eukaryotic ribosomes and chaperones and, at the same time, the <it>E. coli </it>S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.</p

Clinically Actionable Hypercholesterolemia and Hypertriglyceridemia in Children with Nonalcoholic Fatty Liver Disease

OBJECTIVE: To determine the percentage of children with nonalcoholic fatty liver disease (NAFLD) in whom intervention for low-density lipoprotein cholesterol or triglycerides was indicated based on National Heart, Lung, and Blood Institute guidelines. STUDY DESIGN: This multicenter, longitudinal cohort study included children with NAFLD enrolled in the National Institute of Diabetes and Digestive and Kidney Diseases Nonalcoholic Steatohepatitis Clinical Research Network. Fasting lipid profiles were obtained at diagnosis. Standardized dietary recommendations were provided. After 1 year, lipid profiles were repeated and interpreted according to National Heart, Lung, and Blood Institute Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction. Main outcomes were meeting criteria for clinically actionable dyslipidemia at baseline, and either achieving lipid goal at follow-up or meeting criteria for ongoing intervention. RESULTS: There were 585 participants, with a mean age of 12.8 years. The prevalence of children warranting intervention for low-density lipoprotein cholesterol at baseline was 14%. After 1 year of recommended dietary changes, 51% achieved goal low-density lipoprotein cholesterol, 27% qualified for enhanced dietary and lifestyle modifications, and 22% met criteria for pharmacologic intervention. Elevated triglycerides were more prevalent, with 51% meeting criteria for intervention. At 1 year, 25% achieved goal triglycerides with diet and lifestyle changes, 38% met criteria for advanced dietary modifications, and 37% qualified for antihyperlipidemic medications. CONCLUSIONS: More than one-half of children with NAFLD met intervention thresholds for dyslipidemia. Based on the burden of clinically relevant dyslipidemia, lipid screening in children with NAFLD is warranted. Clinicians caring for children with NAFLD should be familiar with lipid management

In Children with Nonalcoholic Fatty Liver Disease, Zone 1 Steatosis is Associated with Advanced Fibrosis

Background & Aims Focal zone 1 steatosis, although rare in adults with nonalcoholic fatty liver disease (NAFLD), does occur in children with NAFLD. We investigated whether focal zone 1 steatosis and focal zone 3 steatosis are distinct subphenotypes of pediatric NAFLD. We aimed to determine associations between the zonality of steatosis and demographic, clinical, and histologic features in children with NAFLD. Methods We performed a cross-sectional study of baseline data from 813 children (age <18 years; mean age, 12.8 ± 2.7 years). The subjects had biopsy-proven NAFLD and were enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network. Liver histology was reviewed using the Nonalcoholic Steatohepatitis Clinical Research Network scoring system. Results Zone 1 steatosis was present in 18% of children with NAFLD (n = 146) and zone 3 steatosis was present in 32% (n = 244). Children with zone 1 steatosis were significantly younger (10 vs 14 years; P < .001) and a significantly higher proportion had any fibrosis (81% vs 51%; P < .001) or advanced fibrosis (13% vs 5%; P < .001) compared with children with zone 3 steatosis. In contrast, children with zone 3 steatosis were significantly more likely to have steatohepatitis (30% vs 6% in children with zone 1 steatosis; P < .001). Conclusions Children with zone 1 or zone 3 distribution of steatosis have an important subphenotype of pediatric NAFLD. Children with zone 1 steatosis are more likely to have advanced fibrosis and children with zone 3 steatosis are more likely to have steatohepatitis. To achieve a comprehensive understanding of pediatric NAFLD, studies of pathophysiology, natural history, and response to treatment should account for the zonality of steatosis

Hepatic Stress Response Mechanisms in Progressive Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients).The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease

Hepatic stress response mechanisms in progressive human nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide, chronic liver disease of increasing clinical significance. It is closely associated with the rising epidemics of obesity and insulin resistance. Up to 17% of the United States population may progress from the disease stage characterized as simple, benign steatosis to the more severe, inflammatory stage of nonalcoholic steatohepatitis (NASH). This progression occurs through 2nd 'hits' of increased oxidative stress and inflammation to a liver that has been sensitized by lipotoxic stress. NASH is also characterized by increased collagen deposition resulting in fibrosis and architectural rearrangement of the liver. Progressive NAFLD is currently recognized as an important contributor to the development of cryptogenic cirrhosis and subsequent liver-related mortalities (estimated at 30-40% in these patients). The pathological progression of NAFLD, as described by the 'two hit' hypothesis, characterizes the different stages of liver injury. However, the mechanism(s) responsible for the progression to NASH are unknown. Profiling global gene expression and metabolite patterns in human liver samples representing the full spectrum of progressive human NAFLD may reveal potential mechanisms of progressive disease. Human liver samples representing each stage of NAFLD progression were analyzed by methodologies such as high-throughput microarrays, high resolution mass spectrometry, and protein immunoblot techniques. Bioinformatics tools and gene expression/regulation database software were utilized in several studies to characterize the altered hepatic profiles of these patients. Hepatic transcriptomic profiles of ADME (absorption, distribution, metabolism and elimination) and ER (endoplasmic reticulum) stress response genes exhibited initiated hepatoprotective responses in patients with NASH. The endogenous pathways of BA (bile acid) synthesis and BCAA (branched chain amino acid) metabolism also showed evidence of coordinately regulated alterations in response to disease-induced stress in NASH. The transcriptional regulation of the investigated pathways was confirmed by transcription factor binding sites enrichment analysis. The collective response to hepatic stress in human NAFLD, demonstrates a coordinated, hepatoprotective intent that may be utilized for future therapeutics in the battle against progressive liver disease

Circulating microRNA 122 in the methionine and choline‐deficient mouse model of non‐alcoholic steatohepatitis

ABSTRACT Non‐alcoholic steatohepatitis (NASH) is a progressive form of non‐alcoholic fatty liver disease (NAFLD) and is a major cause of liver cirrhosis and hepatic failure. The methionine choline‐deficient diet (MCD) is a frequently used hepatotoxicity animal model of NASH that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver‐specific microRNA‐122 (miR‐122) has been shown as a key regulator of cholesterol and fatty acid metabolism in adult liver, and has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. The purpose of this study was to assess miR‐122 serum levels in mice receiving an MCD diet for 0, 3, 7, 14, 28 and 56 days and compare the performance vs. routine clinical chemistry when benchmarked against the histopathological liver findings. MiR‐122 levels were quantified in serum using RT‐qPCR. Both miR‐122 and ALT/AST levels were significantly elevated in serum at all timepoints. MiR‐122 levels increased on average by 40‐fold after 3 days of initiating the MCD diet, whereas ALT and AST changes were 4.8‐ and 3.3‐fold, respectively. In general, miR‐122 levels remained elevated across all time points, whereas the ALT/AST increases were less robust but correlated with the progressive severity of NASH as assessed by histopathology. In conclusion, serum levels of miR‐122 can potentially be used as a sensitive biomarker for the early detection of hepatotoxicity and can aid in monitoring the extent of NAFLD‐associated liver injury in mouse efficacy models. Copyright © 2013 John Wiley & Sons, Ltd. The methionine choline‐deficient diet (MCD) is a frequently used hepatotoxicity animal model of non‐alcoholic steatohepatitis (NASH) that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver‐specific microRNA‐122 (miR‐122) has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. This study shows that miR‐122 serum levels are elevated earlier than transaminases and may be useful as a sensitive biomarker for the early detection of NAFLD‐associated liver injury

Experimental nonalcoholic steatohepatitis increases exposure to simvastatin hydroxy acid by decreasing hepatic organic anion transporting polypeptide expression

Simvastatin (SIM)-induced myopathy is a dose-dependent adverse drug reaction (ADR) that has been reported to occur in 18.2% of patients receiving a 40- to 80-mg dose. The pharmacokinetics of SIM hydroxy acid (SIMA), the bioactive form of SIM, and the occurrence of SIM-induced myopathy are linked to the function of the organic anion transporting polypeptide (Oatp) hepatic uptake transporters. Genetic polymorphisms in SLCO1B1, the gene for human hepatic OATP1B1, cause decreased elimination of SIMA and increased risk of developing myopathy. Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease, and is known to alter drug transporter expression and drug disposition. The purpose of this study was to assess the metabolism and disposition of SIM in a diet-induced rodent model of NASH. Rats were fed a methionine- and choline-deficient diet for 8 weeks to induce NASH and SIM was administered intravenously. Diet-induced NASH caused increased plasma retention and decreased biliary excretion of SIMA due to decreased protein expression of multiple hepatic Oatps. SIM exhibited increased volume of distribution in NASH as evidenced by increased muscle, decreased plasma, and no change in biliary concentrations. Although Cyp3a and Cyp2c11 proteins were decreased in NASH, no alterations in SIM metabolism were observed. These data, in conjunction with our previous data showing that human NASH causes a coordinated downregulation of hepatic uptake transporters, suggest that NASH-mediated transporter regulation may play a role in altered SIMA disposition and the occurrence of myopathy

Modeling human nonalcoholic steatohepatitis-associated changes in drug transporter expression using experimental rodent models

Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents