Publications by authors named "Fraser Duthie"

25 Publications

  • Page 1 of 1

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association.

Commun Biol 2021 02 3;4(1):155. Epub 2021 Feb 3.

University of Sydney, Sydney, New South Wales, 2006, Australia.

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-020-01469-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859232PMC
February 2021

The Detection of Lymphatic Invasion in Colorectal Polyp Cancer Using D2-40 Immunohistochemistry and Its Association With Prognosis.

Cureus 2020 Nov 9;12(11):e11394. Epub 2020 Nov 9.

Department of Pathology, Queen Elizabeth University Hospital, Glasgow, GBR.

Introduction The aim of this study was to compare the detection of lymphatic invasion using haematoxylin and eosin (H&E) staining versus D2-40 immunostaining on specimens from a retrospective cohort of patients with colorectal polyp cancer and to investigate the association of lymphatic invasion, detected by either method, with survival. Methods Specimens from patients with pathologically diagnosed colorectal polyp cancer were selected from the Greater Glasgow and Clyde Bowel Cancer Screening Registry for D2-40 immunohistochemistry staining. Clinicopathological information was retrieved from patient electronic records including analysis of pathology reports to determine if a lymphatic invasion was detected using H&E staining. Results Over 100 patients were included in this study with a median age at polypectomy of 66 years (range 50-76). All patients were followed up for a minimum of four years and five patients died due to colorectal cancer. The lymphatic invasion was detected in 8% of cases by H&E staining and 23% of cases with D2-40 immunostaining. Only D2-40-detected lymphatic invasion showed a statistically significant relationship with colorectal cancer-specific mortality using univariate analysis (p=0.01). Survival analysis performed separately by Cox regression demonstrated that lymphatic invasion detected by D2-40 immunostaining was associated with worse disease-specific survival (hazard ratio [HR] 14.07, 95% CI 1.57-125.97, p=0.018). Conclusion This study shows that D2-40 immunostaining can improve the detection of lymphatic invasion in colorectal polyp cancer when compared to H&E staining. In addition, the lymphatic invasion detected by D2-40 immunostaining significantly associates with survival allowing it to be used as a prognostic indicator in colorectal polyp cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7759/cureus.11394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654984PMC
November 2020

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

Gastroenterology 2021 01 9;160(1):362-377.e13. Epub 2020 Oct 9.

Barts Cancer Institute, Queen Mary University of London, London, United Kingdom.

Background & Aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC.

Methods: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids.

Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency.

Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2020.09.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167930PMC
January 2021

Precision Oncology in Surgery: Patient Selection for Operable Pancreatic Cancer.

Ann Surg 2020 08;272(2):366-376

Department of Surgery, Universitätsklinikum Erlangen, Erlangen, Germany.

Objective: We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection.

Background: Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease.

Method: In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram.

Results: High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables.

Conclusions: Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/SLA.0000000000003143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373491PMC
August 2020

Studying Conformational Changes of the Yersinia Type-III-Secretion Effector YopO in Solution by Integrative Structural Biology.

Structure 2019 09 11;27(9):1416-1426.e3. Epub 2019 Jul 11.

Institute for Physical and Theoretical Chemistry, University of Bonn, 53115 Bonn, Germany. Electronic address:

The type-III secretion effector YopO helps pathogenic Yersinia to outmaneuver the human immune system. Injected into host cells, it functions as a Ser/Thr kinase after activation by actin binding. This activation process is thought to involve large conformational changes. We use PELDOR spectroscopy and small-angle X-ray scattering in combination with available crystal structures to study these conformational transitions. Low-resolution hybrid models of the YopO/actin structure in solution were constructed, where the kinase domain of YopO is tilted "backward" compared with the crystal structure, thus shortening the distance between actin and the kinase active site, potentially affecting the substrate specificity of YopO. Furthermore, the GDI domain of the hybrid models resembles a conformation that was previously observed in a crystal structure of the isolated GDI domain. We investigate possible structural reasons for the inactivity of the apo state, analyze its flexibility and discuss the biological implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2019.06.007DOI Listing
September 2019

Feasibility and clinical utility of endoscopic ultrasound guided biopsy of pancreatic cancer for next-generation molecular profiling.

Chin Clin Oncol 2019 Apr;8(2):16

Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK; West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK.

Next-generation sequencing is enabling molecularly guided therapy for many cancer types, yet failure rates remain relatively high in pancreatic cancer (PC). The aim of this study is to investigate the feasibility of genomic profiling using endoscopic ultrasound (EUS) biopsy samples to facilitate personalised therapy for PC. Ninty-five patients underwent additional research biopsies at the time of diagnostic EUS. Diagnostic formalin-fixed (FFPE) and fresh frozen EUS samples underwent DNA extraction, quantification and targeted gene sequencing. Whole genome (WGS) and RNA sequencing was performed as proof of concept. Only 2 patients (2%) with a diagnosis of PC had insufficient material for targeted sequencing in both FFPE and frozen specimens. Targeted panel sequencing (n=54) revealed mutations in PC genes (KRAS, GNAS, TP53, CDKN2A, SMAD4) in patients with histological evidence of PC, including potentially actionable mutations (BRCA1, BRCA2, ATM, BRAF). WGS (n=5) of EUS samples revealed mutational signatures that are potential biomarkers of therapeutic responsiveness. RNA sequencing (n=35) segregated patients into clinically relevant molecular subtypes based on transcriptome. Integrated multi-omic analysis of PC using standard EUS guided biopsies offers clinical utility to guide personalized therapy and study the molecular pathology in all patients with PC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21037/cco.2019.04.06DOI Listing
April 2019

Non-functional duodenal neuroendocrine carcinoma: a rare cause of diabetes mellitus.

Endocrinol Diabetes Metab Case Rep 2018 23;2018. Epub 2018 Aug 23.

NHS Ayrshire and Arran, Ayr, UK.

A 40-year-old Caucasian female presented with hyperglycaemia, polyuria, polydipsia and weight loss of 6 kg over a 1-month period. There was no personal or family history of malignancy or diabetes mellitus. On examination, she was jaundiced with pale mucous membranes and capillary glucose was 23.1 mmol/L. Initial investigations showed iron deficiency anaemia and obstructive pattern of liver function tests. HbA1c was diagnostic of diabetes mellitus at 79 mmol/mol. Malignancy was suspected and CT chest, abdomen and pelvis showed significant dilatation of intra- and extra-hepatic biliary tree including pancreatic duct, with periampullary 30 mm mass lesion projecting into lumen of duodenum. Enlarged nodes were seen around the superior mesenteric artery. This was confirmed on MRI liver. Fasting gut hormones were normal except for a mildly elevated somatostatin level. Chromogranin A was elevated at 78 pmol/L with normal chromogranin B. Duodenoscopy and biopsy showed possible tubovillous adenoma with low-grade dysplasia, but subsequent endoscopic ultrasound and biopsy revealed a grade 1, well differentiated neuroendocrine tumour. The patient was started on insulin, transfused to Hb >8 g/dL and Whipple's pancreatico-duodenectomy was undertaken. This showed a well-differentiated neuroendocrine carcinoma arising in duodenum (Grade G1 with Ki67: 0.5%), with areas of chronic pancreatitis and preservation of pancreatic islet cells. There was complete resolution of diabetes post Whipple's procedure and patient was able to come of insulin treatment. Her last HBA1C was 31 mmol/mol, 4 months post tumour resection.

Learning Points: Diabetes mellitus and malignancy can be related.A high index of suspicion is needed when diabetes mellitus presents atypically.Non-functional neuroendocrine tumours can present with diabetes mellitus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/EDM-18-0061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109206PMC
August 2018

The role of induction chemotherapy + chemoradiotherapy in localised pancreatic cancer: initial experience in Scotland.

J Gastrointest Oncol 2017 Aug;8(4):683-695

Beatson West of Scotland Cancer Centre, Glasgow, UK.

Background: Despite being relatively rare pancreatic cancer is one of the highest causes of death. Even within the potentially resectable group outcomes are poor. We present our initial experiences utilising a neoadjuvant approach to localised pancreatic cancer, evaluating survival, response rates and tolerability.

Methods: This was a retrospective analysis of a prospectively maintained database. Patients from 2012 to 2015 referred to a busy regional Hepato-Pancreatic Biliary (HPB) MDT were included. Patients were classified according to respectability criteria (utilising NCCN guidelines) and a treatment plan agreed. Systemic therapy with either FOLFIRINOX or Gem/Cap was delivered followed by chemoradiotherapy if disease remained localised. Toxicity, response, pathological outcomes and survival were all recorded.

Results: A total of 85 patients were included in the study: 45 had initially resectable disease; 19 required a response for resection and 21 had locally advanced inoperable disease; 34 patients underwent resection. The median survival for the potentially resectable group was 22.2 months while for those undergoing resection it was 37 months.

Conclusions: We have demonstrated that a neoadjuvant approach is deliverable and tolerable. In addition we have demonstrated impressive survival results in patients undergoing resection with no detriment in outcome for those not proceeding to surgery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21037/jgo.2017.04.01DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582039PMC
August 2017

Whole-genome landscape of pancreatic neuroendocrine tumours.

Nature 2017 03 15;543(7643):65-71. Epub 2017 Feb 15.

QIMR Berghofer Medical Research Institute, Herston Road, Brisbane 4006, Australia.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature21063DOI Listing
March 2017

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells.

Angew Chem Int Ed Engl 2017 01 5;56(1):177-181. Epub 2016 Dec 5.

Institute of Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115, Bonn, Germany.

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201609085DOI Listing
January 2017

Hypermutation In Pancreatic Cancer.

Gastroenterology 2017 01 15;152(1):68-74.e2. Epub 2016 Nov 15.

QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2016.09.060DOI Listing
January 2017

Studies on the X-Ray and Solution Structure of FeoB from Escherichia coli BL21.

Biophys J 2016 Jun;110(12):2642-2650

Institute for Physical and Theoretical Chemistry,University of Bonn, Bonn, Germany.

The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bpj.2016.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919421PMC
June 2016

A Glasgow Tipple-transjugular intrahepatic portosystemic shunt insertion prior to Whipple resection.

J Surg Case Rep 2016 May 13;2016(5). Epub 2016 May 13.

West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK.

Abdominal surgery performed in patients with significant liver disease and portal hypertension is associated with high mortality rates, with even poorer outcomes associated with complex pancreaticobiliary operations. We report on a patient requiring portal decompression via transjugular intrahepatic portosystemic shunt (TIPS) prior to a pancreaticoduodenectomy. The 49-year-old patient presented with pain, jaundice and weight loss. At ERCP an edematous ampulla was biopsied, revealing high-grade dysplasia within a distal bile duct adenoma. Liver biopsy was performed to investigate portal hypertension, confirming congenital hepatic fibrosis (CHF). A TIPS was performed to enable a pancreaticoduodenectomy. Prophylactic TIPS can be performed for preoperative portal decompression for patients requiring pancreatic resection. A potentially curative resection was performed when abdominal surgery was initially thought impossible. Notably, CHF has been associated with the development of cholangiocarcinoma in only four previous instances, with this case being only the second reported distal bile duct cholangiocarcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jscr/rjw089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866079PMC
May 2016

Investigating Various Thresholds as Immunohistochemistry Cutoffs for Observer Agreement.

Appl Immunohistochem Mol Morphol 2017 Oct;25(9):599-608

*Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow §Institute of Cardiovascular and Medical Sciences, University of Glasgow, Western Infirmary ¶Academic Unit of Surgery, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow Royal Infirmary ‡Department of Pathology, Laboratory Medicine Building, Queen Elizabeth University Hospital, Greater Glasgow & Clyde NHS ∥West of Scotland Pancreatic Unit and Glasgow Royal Infirmary, Alexandra Parade, Glasgow #Pathology Laboratory, Forth Valley Royal Hospital, Larbert, UK †Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan.

Background: Clinical translation of immunohistochemistry (IHC) biomarkers requires reliable and reproducible cutoffs or thresholds for interpretation of immunostaining. Most IHC biomarker research focuses on the clinical relevance (diagnostic, prognostic, or predictive utility) of cutoffs, with less emphasis on observer agreement using these cutoffs. From the literature, we identified 3 commonly used cutoffs of 10% positive epithelial cells, 20% positive epithelial cells, and moderate to strong staining intensity (+2/+3 hereafter) to use for investigating observer agreement.

Materials And Methods: A series of 36 images of microarray cores stained for 4 different IHC biomarkers, with variable staining intensity and percentage of positive cells, was used for investigating interobserver and intraobserver agreement. Seven pathologists scored the immunostaining in each image using the 3 cutoffs for positive and negative staining. Kappa (κ) statistic was used to assess the strength of agreement for each cutoff.

Results: The interobserver agreement between all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.64, 0.59, and 0.62, respectively, for 10%, 20%, and +2/+3 cutoffs. A good agreement was observed for experienced pathologists using the 10% cutoff, and their agreement was statistically higher than for junior pathologists (P=0.02). In addition, the mean intraobserver agreement for all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.71, 0.60, and 0.73, respectively, for 10%, 20%, and +2/+3 cutoffs. For all 3 cutoffs, a positive correlation was observed with perceived ease of interpretation (P<0.003). Finally, cytoplasmic-only staining achieved higher agreement using all 3 cutoffs than mixed staining patterns.

Conclusions: All 3 cutoffs investigated achieve reasonable strength of agreement, modestly decreasing interobserver and intraobserver variability in IHC interpretation. These cutoffs have previously been used in cancer pathology, and this study provides evidence that these cutoffs can be reproducible between practicing pathologists.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/PAI.0000000000000357DOI Listing
October 2017

Genomic analyses identify molecular subtypes of pancreatic cancer.

Nature 2016 Mar 24;531(7592):47-52. Epub 2016 Feb 24.

Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature16965DOI Listing
March 2016

Ampullary Cancers Harbor ELF3 Tumor Suppressor Gene Mutations and Exhibit Frequent WNT Dysregulation.

Cell Rep 2016 Feb 21;14(4):907-919. Epub 2016 Jan 21.

Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia; Department of Surgery, Royal North Shore Hospital, St Leonards, Sydney, NSW 2065, Australia.

The ampulla of Vater is a complex cellular environment from which adenocarcinomas arise to form a group of histopathologically heterogenous tumors. To evaluate the molecular features of these tumors, 98 ampullary adenocarcinomas were evaluated and compared to 44 distal bile duct and 18 duodenal adenocarcinomas. Genomic analyses revealed mutations in the WNT signaling pathway among half of the patients and in all three adenocarcinomas irrespective of their origin and histological morphology. These tumors were characterized by a high frequency of inactivating mutations of ELF3, a high rate of microsatellite instability, and common focal deletions and amplifications, suggesting common attributes in the molecular pathogenesis are at play in these tumors. The high frequency of WNT pathway activating mutation, coupled with small-molecule inhibitors of β-catenin in clinical trials, suggests future treatment decisions for these patients may be guided by genomic analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982376PMC
February 2016

Expression, purification and spin labelling of the ferrous iron transporter FeoB from Escherichia coli BL21 for EPR studies.

Protein Expr Purif 2015 Oct 9;114:30-6. Epub 2015 Jun 9.

Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany.

The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2015.05.014DOI Listing
October 2015

Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM).

J Inorg Biochem 2015 Jul 22;148:49-56. Epub 2015 May 22.

Institute of Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, D-53115 Bonn, Germany. Electronic address:

The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine-Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV-Visible (UV-Vis) spectroscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jinorgbio.2015.05.008DOI Listing
July 2015

Comparison of PELDOR and RIDME for Distance Measurements between Nitroxides and Low-Spin Fe(III) Ions.

J Phys Chem B 2015 Oct 3;119(43):13534-42. Epub 2015 Jun 3.

Institute of Physical and Theoretical Chemistry, University of Bonn , Wegelerstrasse 12, 53115 Bonn, Germany.

EPR-based nanometre distance measurements are becoming ever more important in structural biology. Usually the distance constraints are measured between two nitroxide spin labels. Yet, distance measurements between a metal center and spin labels enable, e.g., the localization of metal ions within the tertiary fold of biomolecules. Therefore, it is important to find methods that provide such distance information quickly, with high precision and reliability. In the present study, two methods, pulsed electron-electron double resonance (PELDOR) and relaxation-induced dipolar modulation enhancement (RIDME), are compared on the heme-containing and spin-labeled cytochrome P450cam. Special emphasis is put on the optimization of the dead-time free RIDME experiment and several ways of data analysis. It turned out that RIDME appears to be better suited for distance measurements involving metal ions like low-spin Fe(3+) than PELDOR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jpcb.5b02118DOI Listing
October 2015

Expression of KOC, S100P, mesothelin and MUC1 in pancreatico-biliary adenocarcinomas: development and utility of a potential diagnostic immunohistochemistry panel.

BMC Clin Pathol 2014 23;14:35. Epub 2014 Jul 23.

Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, College of Medical Veterinary and Life Sciences, University of Glasgow, Garscube Estate, Switchback Road, Bearsden G61 1QH, UK ; Department of Pathology, Southern General Hospital, Greater Glasgow & Clyde NHS, Glasgow G51 4TF, UK.

Background: Pancreatico-biliary adenocarcinomas (PBA) have a poor prognosis. Diagnosis is usually achieved by imaging and/or endoscopy with confirmatory cytology. Cytological interpretation can be difficult especially in the setting of chronic pancreatitis/cholangitis. Immunohistochemistry (IHC) biomarkers could act as an adjunct to cytology to improve the diagnosis. Thus, we performed a meta-analysis and selected KOC, S100P, mesothelin and MUC1 for further validation in PBA resection specimens.

Methods: Tissue microarrays containing tumour and normal cores in a ratio of 3:2, from 99 surgically resected PBA patients, were used for IHC. IHC was performed on an automated platform using antibodies against KOC, S100P, mesothelin and MUC1. Tissue cores were scored for staining intensity and proportion of tissue stained using a Histoscore method (range, 0-300). Sensitivity and specificity for individual biomarkers, as well as biomarker panels, were determined with different cut-offs for positivity and compared by summary receiver operating characteristic (ROC) curve.

Results: The expression of all four biomarkers was high in PBA versus normal ducts, with a mean Histoscore of 150 vs. 0.4 for KOC, 165 vs. 0.3 for S100P, 115 vs. 0.5 for mesothelin and 200 vs. 14 for MUC1 (p < .0001 for all comparisons). Five cut-offs were carefully chosen for sensitivity/specificity analysis. Four of these cut-offs, namely 5%, 10% or 20% positive cells and Histoscore 20 were identified using ROC curve analysis and the fifth cut-off was moderate-strong staining intensity. Using 20% positive cells as a cut-off achieved higher sensitivity/specificity values: KOC 84%/100%; S100P 83%/100%; mesothelin 88%/92%; and MUC1 89%/63%. Analysis of a panel of KOC, S100P and mesothelin achieved 100% sensitivity and 99% specificity if at least 2 biomarkers were positive for 10% cut-off; and 100% sensitivity and specificity for 20% cut-off.

Conclusion: A biomarker panel of KOC, S100P and mesothelin with at least 2 biomarkers positive was found to be an optimum panel with both 10% and 20% cut-offs in resection specimens from patients with PBA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6890-14-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112611PMC
July 2014

The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Jan 25;69(Pt 1):25-34. Epub 2012 Dec 25.

Biomedical Sciences Research Complex, University of St Andrews, St Andrews KY16 9ST, Scotland.

Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1744309112044739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539698PMC
January 2013

Structural insights into the mechanism and inhibition of the β-hydroxydecanoyl-acyl carrier protein dehydratase from Pseudomonas aeruginosa.

J Mol Biol 2013 Jan 19;425(2):365-77. Epub 2012 Nov 19.

Biomedical Sciences Research Complex, The University, St. Andrews KY16 9ST, UK.

Fatty acid biosynthesis is an essential component of metabolism in both eukaryotes and prokaryotes. The fatty acid biosynthetic pathway of Gram-negative bacteria is an established therapeutic target. Two homologous enzymes FabA and FabZ catalyze a key step in fatty acid biosynthesis; both dehydrate hydroxyacyl fatty acids that are coupled via a phosphopantetheine to an acyl carrier protein (ACP). The resulting trans-2-enoyl-ACP is further polymerized in a processive manner. FabA, however, carries out a second reaction involving isomerization of trans-2-enoyl fatty acid to cis-3-enoyl fatty acid. We have solved the structure of Pseudomonas aeruginosa FabA with a substrate allowing detailed molecular insight into the interactions of the active site. This has allowed a detailed examination of the factors governing the second catalytic step. We have also determined the structure of FabA in complex with small molecules (so-called fragments). These small molecules occupy distinct regions of the active site and form the basis for a rational inhibitor design program.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2012.11.017DOI Listing
January 2013

Endoscopic ultrasound-guided elastography in the nodal staging of oesophageal cancer.

World J Gastroenterol 2012 Mar;18(9):889-95

Department of Gastroenterology, Forth Valley Royal Hospital, Larbert FK5 4WR, United Kingdom.

Aim: To assess quantitative endoscopic ultrasound (EUS)-guided elastography in the nodal staging of oesophago-gastric cancers.

Methods: This was a single tertiary centre study assessing 50 patients with established oesophago-gastric cancer undergoing EUS-guided fine needle aspiration biopsy (FNAB) of lymph nodes between July 2007 and July 2009. EUS-guided elastography of lymph nodes was performed before EUS-FNAB. Standard EUS characteristics were also described. Cytological determination of whether a lymph node was malignant or benign was used as the gold standard for this study. Comparisons of elastography and standard EUS characteristics were made between the cytologically benign and malignant nodes. The main outcome measure was the accuracy of elastography in differentiating between benign and malignant lymph nodes in oesophageal cancers.

Results: EUS elastography and FNAB were performed on 53 lymph nodes. Cytological malignancy was found in 23 nodes, one was indeterminate, one was found to be a gastrointestinal stromal tumor and 25 of the nodes were negative for malignancy. On 3 occasions insufficient material was obtained for analysis. The area under the curve for the receiver operating characteristic curve for elastography strain ratio was 0.87 (P < 0.0001). Elastography strain ratio had a sensitivity 83%, specificity 96%, positive predictive value 95%, and negative predictive value 86% for distinguishing between malignant and benign nodes. The overall accuracy of elastography strain ratio was 90%. Elastography was more sensitive and specific in determining malignant nodal disease than standard EUS criteria.

Conclusion: EUS elastography is a promising modality that may complement standard EUS and help guide EUS-FNAB during staging of upper gastrointestinal tract cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3748/wjg.v18.i9.889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297047PMC
March 2012

A multi-centre pathologist survey on pathological processing and regression grading of colorectal cancer resection specimens treated by neoadjuvant chemoradiation.

Virchows Arch 2012 Feb 13;460(2):151-5. Epub 2012 Jan 13.

Department of Cellular Pathology, John Radcliffe Hospital and University of Oxford, Oxford, UK.

To ascertain the approach and degree of consensus of pathologists in the handling and regression grading of colorectal cancer resection specimens treated with neoadjuvant chemoradiation, a ten-part questionnaire was circulated to 18 gastrointestinal pathologists in eight countries. The questions were specific and addressed pertinent issues related to colorectal cancer with neoadjuvant chemoradiation. There is a lack of consensus on how to handle the specimen, number of sections taken, correlation with pre- and post-operative radiological imaging, and especially, regression grading schema employed. Consensus in the form of guidelines is required so that the pathological assessment of these specimens will provide clinically relevant information for patient management, irrespective of location.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00428-012-1193-3DOI Listing
February 2012
-->