Jeremy French

Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.

BackgroundChronic liver injury can lead to the development of liver fibrosis and cirrhosis but only in a minority of patients. Currently, it is not clear which factors determine progression to fibrosis. We investigated whether DNA\methylation profile as determined by pyrosequencing can distinguish patients with mild from those with advanced/severe fibrosis in non-alcoholic liver disease (NAFLD) and alcoholic liver disease (ALD). To this end, paraffin-embedded liver biopsies were collected from patients with biopsy-proven NAFLD or ALD, as well as paraffin-embedded normal liver resections, genomic DNA isolated, bisulfite converted and pyrosequencing assays used to quantify DNA methylation at specific CpGs within PPARα, PPARα, TGFβ1, Collagen 1A1 and PDGFα genes. Furthermore, we assessed the impact of age, gender and anatomical location within the liver on patterns of DNA methylation in the same panel of genes.ResultsDNA methylation at specific CpGs within genes known to affect fibrogenesis distinguishes between patients with mild from those with severe fibrosis in both NAFLD and ALD, although same CpGs are not equally represented in both etiologies. In normal liver, age, gender or anatomical location had no significant impact on DNA methylation patterns in the liver.ConclusionsDNA methylation status at specific CpGs may be useful as part of a wider set of patient data for predicting progression to liver fibrosis.

Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease

Objective Liver biopsy is currently the most reliable way of evaluating liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). Its inherent risks limit its widespread use. Differential liver DNA methylation of peroxisome proliferator-activated receptor gamma (PPARγ) gene promoter has recently been shown to stratify patients in terms of fibrosis severity but requires access to liver tissue. The aim of this study was to assess whether DNA methylation of circulating DNA could be detected in human plasma and potentially used to stratify liver fibrosis severity in patients with NAFLD. Design Patients with biopsy-proven NAFLD and age-matched controls were recruited from the liver and gastroenterology clinics at the Newcastle upon Tyne Hospitals NHS Foundation Trust. Plasma cell-free circulating DNA methylation of PPARγ was quantitatively assessed by pyrosequencing. Liver DNA methylation was quantitatively assessed by pyrosequencing NAFLD explant tissue, subjected to laser capture microdissection (LCM). Patients with alcoholic liver disease (ALD) were also subjected to plasma DNA and LCM pyrosequencing. Results 26 patients with biopsy-proven NAFLD were included. Quantitative plasma DNA methylation of PPARγ stratified patients into mild (Kleiner 1–2) and severe (Kleiner 3–4) fibrosis (CpG1: 63% vs 86%, p<0.05; CpG2: 51% vs 65% p>0.05). Hypermethylation at the PPARγ promoter of plasma DNA correlated with changes in hepatocellular rather than myofibroblast DNA methylation. Similar results were demonstrated in patients with ALD cirrhosis. Conclusions Differential DNA methylation at the PPARγ promoter can be detected within the pool of cell-free DNA of human plasma. With further validation, plasma DNA methylation of PPARγ could potentially be used to non-invasively stratify liver fibrosis severity in patients with NAFLD. Plasma DNA methylation signatures reflect the molecular pathology associated with fibrotic liver disease.

Alcohol directly stimulates epigenetic modifications in hepatic stellate cells.

BACKGROUND & AIMS Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. METHODS HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patients liver and through precision cut liver slices. RESULTS HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. CONCLUSIONS Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.

Acute pancreatitis and the influence of socioeconomic deprivation

A comprehensive epidemiological study of acute pancreatitis (AP) using reliable objective methods of patient identification with the inclusion of socioeconomic factors has not been reported previously.

Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation landscape

BACKGROUND & AIMS DNA methylation (5-mC) is an epigenetic mark that is an established regulator of transcriptional repression with an important role in liver fibrosis. Currently, there is very little knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cell (HSC), the key cell type responsible for onset and progression of liver fibrosis. Moreover, recently discovered DNA hydroxymethylation (5-hmC) is involved in transcriptional activation and its patterns are often altered in human diseases. The aim of this study is to investigate the role of DNA methylation/hydroxymethylation in liver fibrosis. METHODS Levels of 5-mC and 5-hmC were assessed by slot blot in a range of animal liver fibrosis models and human liver diseases. Expression levels of TET and DNMT enzymes were measured by qRT-PCR and Western blotting. Reduced representation bisulfite sequencing (RRBS) method was used to examine 5-mC and 5-hmC patterns in quiescent and in vivo activated rat HSC. RESULTS We demonstrate global alteration in 5-mC and 5-hmC and their regulatory enzymes that accompany liver fibrosis and HSC transdifferentiation. Using RRBS, we show exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. In addition, we demonstrate that reduction in DNMT3a expression leads to attenuation of pro-fibrogenic phenotype in activated HSC. CONCLUSIONS Our data suggest that DNA 5-mC/5-hmC is a crucial step in HSC activation and therefore fibrogenesis. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.

Systematic review of irreversible electroporation in the treatment of advanced pancreatic cancer

BACKGROUND Irreversible electroporation (IRE) is a novel procedure to combat pancreatic cancer, whereby high voltage pulses are delivered, resulting in cell death. This represents an ideal alternative to other thermal treatment modalities, as there is no overriding heat effect, therefore reducing the risk of injury to vessels and ducts. METHODS Multiple databases were searched to January 2014. Primary outcome measures were survival and associated morbidity. 41 articles were initially identified; of these 4 studies met the inclusion criteria, yielding 74 patients in total. RESULTS 94.5% of patients had locally advanced tumours, the remainder had metastatic disease. Treated tumour size ranged from 1 to 7 cm. IRE approach included open (70.3%), laparoscopic (2.7%) and percutaneous (27%; ultrasound-guided 30%, CT-guided 70%) Morbidity ranged from 0 to 33%; due to the high number of simultaneous procedures performed (resection/bypass) it was difficult to ascertain IRE-related complications. However no significant bleeding occurred when IRE-alone was performed. Survival statistics suggest a prognostic benefit. Reported survival included: 6 month survival of 40% (n = 5) and 70% (n = 14); PFS and OS 14 and 20 months respectively (n = 54). Results of most interest showed a significant survival benefit in matched IRE vs non-IRE groups (PFS 14 vs 6 mths; p = 0.01, OS 20 vs 11 mths; p = 0.03). CONCLUSION Initial evidence suggests IRE incurs a prognostic benefit with minimal morbidity. More high quality research is required to determine the role IRE may play in the multi-modal management of pancreatic cancers.

Metastatic lymph node ratio as an important prognostic factor in pancreatic ductal adenocarcinoma.

BACKGROUND Overall five year survival following pancreaticoduodenectomy for ductal adenocarcinoma is poor with typical reported rates in the literature of 8-27%. The aim of this study was to identify the histological variables best able to predict long-term survival in these patients. METHODS A prospective database of patients undergoing pancreaticoduodenectomy between April 2002 and June 2009 was analysed to identify patients with histologically proven pancreatic ductal adenocarcinoma. Patients with ampullary tumours, cholangiocarcinoma, duodenal adenocarcinoma and neuroendocrine tumours were excluded. The histology reports for these patients were reviewed. Uni-variate and multi-variate survival analysis was performed to identify variables useful in predicting long-term outcome. RESULTS 134 patients underwent pancreaticoduodenectomy for pancreatic ductal adenocarcinoma during this period. 5 year survival in this series was 18.6%. Uni-variate analysis identified nodal status and the metastatic to resected lymph node ratio as predictors of survival. Using multi-variate Cox Regression analysis a metastatic to lymph node ratio of >15% (p < 0.01) and the presence of perineural invasion (p < 0.05) were identified as independent predictors of patient survival. Metastatic to resected lymph node ratio is better able to stratify prognosis than nodal status alone with 5 year survival of those with N0 disease being 55.6% and 12.9% for N1 disease. However for those with <15% of resected nodes positive, 5 year survival was 21.7% and in those with >15% nodes positive it was 5.2% (p = 0.0017). CONCLUSION The metastatic to resected lymph node ratio can provide significant prognostic information in those patients with node positive disease after pancreaticoduodenectomy for pancreatic ductal adenocarcinoma.

Effects of low cardiopulmonary reserve on pancreatic leak following pancreaticoduodenectomy

Postoperative complications are increased in patients with reduced cardiopulmonary reserve undergoing major surgery. Pancreatic leak is an important contributor to postoperative complications and death following pancreaticoduodenectomy. The aim of this study was to determine whether reduced cardiopulmonary reserve was a risk factor for pancreatic leak.

Laparoscopic Liver Resection for Hepatic Adenoma in Pregnancy

Introduction Liver cell adenomas (LCA) can present during pregnancy with abdominal pain and bleeding. Assessment and management at this time are complicated by concerns over fetal well-being. Methods We reviewed cases from our own institution, including the only laparoscopic liver resection reported in pregnancy, and systematically reviewed the literature to identify successful management strategies for this clinical dilemma. Results Two cases of surgery for bleeding liver adenoma were identified in our own institution. One case was managed with an elective laparoscopic segmental resection at 16 weeks and 1 with open surgery and successful fetal delivery at 32 weeks gestation. In the second case hepatic rupture of a 3.5-cm lesion was precipitated by diagnostic biopsy. In the world literature, spontaneous rupture of an LCA during pregnancy has been reported in 19 cases and is associated with maternal mortality and fetal loss approaching 50%. Conclusions We advocate an aggressive approach to management of LCA in pregnancy owing to the high mortality associated with rupture. Biopsy of LCA in pregnancy is unsafe and can be complicated by rupture. Hence, patients presenting de novo with clinical or radiologic signs of bleeding or large (>5 cm) undiagnosed lesions should be offered laparoscopic resection if feasible.

Surgery for retroperitoneal soft tissue sarcomas: aggressive re-resection of recurrent disease is possible

INTRODUCTION Retroperitoneal soft tissue sarcomas represent a relatively rare and complex therapeutic problem where surgery forms the mainstay of treatment and is technically demanding. In this study, we review a single UK centres experience with the surgical management of retro-peritoneal soft tissue sarcoma. PATIENTS AND METHODS We present analysis of data on patients treated between 1997 and 2006, our first 75 patients. Data collected from the Access database, included patient demographics, staging modalities, peri-operative details, treatment, outcome, pathological diagnosis and subsequent complications. RESULTS A total of 75 patients (M:F, 44:31) underwent 115 resectional procedures as part of the management of retroperitoneal soft-tissue sarcoma. There were 12 major complications for the 115 procedures (morbidity of 8.69%). The 30-day operative mortality was zero and the 90-day mortality rate was 1.33% (1/75). Follow-up ranged from 16-131 months. The median disease-free survival was 69 months (range, 59-78 months). Recurrences developed in 46 patients; median time to overall recurrence was 13 months (range, 3-71 months). Of these 46, 22 developed localised recurrence, which was amenable to further resection. In the cohort of patients with recurrent disease, median survival in those who underwent surgery was 53 months (range, 30-76 months) and median survival in those who did not undergo surgery was 30 months (range, 18-41 months) and this difference was statistically significant (log rank, P = 0.01). CONCLUSIONS Extensive resectional surgery with minimal morbidity, devoid of mortality is feasible in the treatment of retroperitoneal sarcoma. Development of recurrent disease is a significant factor influencing survival; however, localised recurrences are amenable to surgery and this can lead to improved survival.