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Low-cost and clinically applicable copy number profiling using repeat DNA.
BACKGROUND: Somatic copy number alterations (SCNAs) are an important class of genomic alteration in cancer. They are frequently observed in cancer samples, with studies showing that, on average, SCNAs affect 34% of a cancer cell's genome. Furthermore, SCNAs have been shown to be major drivers of tumour development and have been associated with response to therapy and prognosis. Large-scale cancer genome studies suggest that tumours are driven by somatic copy number alterations (SCNAs) or single-nucleotide variants (SNVs). Despite the frequency of SCNAs and their clinical relevance, the use of genomics assays in the clinic is biased towards targeted gene panels, which identify SNVs but provide limited scope to detect SCNAs throughout the genome. There is a need for a comparably low-cost and simple method for high-resolution SCNA profiling. RESULTS: We present conliga, a fully probabilistic method that infers SCNA profiles from a low-cost, simple, and clinically-relevant assay (FAST-SeqS). When applied to 11 high-purity oesophageal adenocarcinoma samples, we obtain good agreement (Spearman's rank correlation coefficient, rs=0.94) between conliga's inferred SCNA profiles using FAST-SeqS data (approximately £14 per sample) and those inferred by ASCAT using high-coverage WGS (gold-standard). We find that conliga outperforms CNVkit (rs=0.89), also applied to FAST-SeqS data, and is comparable to QDNAseq (rs=0.96) applied to low-coverage WGS, which is approximately four-fold more expensive, more laborious and less clinically-relevant. By performing an in silico dilution series experiment, we find that conliga is particularly suited to detecting SCNAs in low tumour purity samples. At two million reads per sample, conliga is able to detect SCNAs in all nine samples at 3% tumour purity and as low as 0.5% purity in one sample. Crucially, we show that conliga's hidden state information can be used to decide when a sample is abnormal or normal, whereas CNVkit and QDNAseq cannot provide this critical information. CONCLUSIONS: We show that conliga provides high-resolution SCNA profiles using a convenient, low-cost assay. We believe conliga makes FAST-SeqS a more clinically valuable assay as well as a useful research tool, enabling inexpensive and fast copy number profiling of pre-malignant and cancer samples.
MMP2 and MMP7 at the invasive front of gastric cancer are not associated with mTOR expression.
BACKGROUND: Regulation of MMP expression by activation of mTOR signalling has been demonstrated for several tumor types, but has thus far not been confirmed in gastric cancer. FINDINGS: The study compromised 128 patients who underwent gastric resection for cancer (66.4 % male; 86 intestinal, 42 diffuse type). Immunohistochemical staining of MMPs was performed to analyse the topographical pattern of MMP expression at the tumor center and the invasive front, respectively. MMP2 showed higher expression at the invasive front compared to the tumor center, whereas MMP7 staining scores were higher in the tumor center, and there was no difference for MMP9. The expression of p-mTOR was higher in the tumor center than at the invasive front, with a similar trend for mTOR. For intestinal type gastric cancer there was a weak correlation of MMP9 with expression of mTOR in the tumor center. Otherwise, there was no correlation of the MMPs with mTOR. By treatment of MKN45 gastric cancer cells with rapamycin, a reduction of p-mTOR in the Western blot was achieved; however, expression of MMPs remained unaffected. CONCLUSIONS: Expression of MMP2 and MMP7 in gastric cancer is not associated with mTOR, MMP9 expression might be related to mTOR signalling in a subset of tumors.
Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.
New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.
Multicentre cohort study to define and validate pathological assessment of response to neoadjuvant therapy in oesophagogastric adenocarcinoma.
BACKGROUND: This multicentre cohort study sought to define a robust pathological indicator of clinically meaningful response to neoadjuvant chemotherapy in oesophageal adenocarcinoma. METHODS: A questionnaire was distributed to 11 UK upper gastrointestinal cancer centres to determine the use of assessment of response to neoadjuvant chemotherapy. Records of consecutive patients undergoing oesophagogastric resection at seven centres between January 2000 and December 2013 were reviewed. Pathological response to neoadjuvant chemotherapy was assessed using the Mandard Tumour Regression Grade (TRG) and lymph node downstaging. RESULTS: TRG (8 of 11 centres) was the most widely used system to assess response to neoadjuvant chemotherapy, but there was discordance on how it was used in practice. Of 1392 patients, 1293 had TRG assessment; data were available for clinical and pathological nodal status (cN and pN) in 981 patients, and TRG, cN and pN in 885. There was a significant difference in survival between responders (TRG 1-2; median overall survival (OS) not reached) and non-responders (TRG 3-5; median OS 2·22 (95 per cent c.i. 1·94 to 2·51) years; P
Gastric MALT lymphoma - update on diagnosis and treatment.
Gastrointestinal lymphoma represent a heterogenous group with differences in pathogenesis, treatment and prognosis. Gastric MALT lymphoma is the most common entity. Helicobacter pylori has been identified as its decisive pathogenetic factor. Once a definitive diagnosis has been established a staging procedure is obligatory for defining the stage of disease. H. pylori eradication is the treatment of choice in all MALT lymphoma patients being infected by the bacterium. In some 70-80% of patients with stages I/II complete regression of the lymphoma will develop after successful eradication of H. pylori. Another 20% of patients will reveal minimal histological residuals after eradication. They can be successfully managed by a watch-and-wait strategy if initial endoscopic abnormalities disappear. At present, it is unclear if this strategy can be also offered to patients with persisting minimal endoscopic abnormalities. Why eradication therapy is effective in some patients with negative H. pylori status is highly speculative at present. Non-responders to H. pylori therapy are transferrred to radiotherapy in stages I/II or to immuno-chemotherapy in stages III/IV.
Gastric acid secretion: changes during a century.
The advances in knowledge of gastric physiology within the past century have been the most exciting and important in this area of interest for many decades. The aim of this presentation consists of a comprehensive review of the extensive recent literature on this topic in order to highlight milestones in the field of gastric physiology, in particular in gastric acid secretion, gastric pathophysiology, acid-related diseases and use of acid regulatory drugs. Moreover, in the 21st century there have been many epidemiologic changes as well as a decrease of Helicobacter pylori infection and gastric cancer together with an increase of gastroesophageal reflux disease and the related increase of pomp proton inhibitor wide use.
Implementation of gastric cancer screening - the global experience.
Gastric cancer (GC) is still an important global healthcare problem, and in absolute figures it is going to remain at the present level in foreseeable future. In general, survival of patients with GC is poor mainly due to advanced-stage diagnosis. Early-stage GC can be cured by endoscopic resection or less invasive surgical treatment. Unfortunately, there is no appropriate screening strategy available for global application. This article provides a description of established national and regional GC screening programs and the screening modalities used. This review also summarizes current approaches to develop cancer-screening biomarkers. Although candidates with initial promising results have been suggested, moving discovery into clinical practice is still a major challenge. Well-designed biomarker studies, with systematic validation steps, are needed to decrease the burden of this fatal disease.
Clinical relevance of Helicobacter pylori vacA and cagA genotypes in gastric carcinoma.
Helicobacter pylori infection is the major etiological factor of gastric carcinoma. This disease is the result of a long, multistep, and multifactorial process, which occurs only in a small proportion of patients infected with H. pylori. Gastric carcinoma development is influenced by host genetic susceptibility factors, environmental factors, and H. pylori virulence. H. pylori is genetically highly variable, and variability that affects H. pylori virulence factors may be useful to identify strains with different degrees of pathogenicity. This review will focus on VacA and CagA that have polymorphic regions that impact their functional properties. The characterization of H. pylori vacA and cagA-associated could be useful for identifying patients at highest risk of disease, who could be offered H. pylori eradication therapy and who could be included in programs of more intensive surveillance in an attempt to reduce gastric carcinoma incidence.
Chronic gastritis - an update.
Helicobacter pylori is the main aetiologic factor for chronic gastritis worldwide. The degree of inflammation and the evolution of this form of chronic gastritis can vary largely depending on bacterial virulence factors, host susceptibility factors and environmental conditions. Autoimmune gastritis is another cause of chronic inflammation in the stomach, which can occur in all age groups. This disease presents typically with vitamin B12 deficiency and pernicious anaemia. The presence of anti-parietal cell antibodies is highly specific for the diagnosis. The role of H. pylori as a trigger for autoimmune gastritis remains uncertain. Other rare conditions for chronic gastritis are chronic inflammatory conditions such as Crohn's disease or on the background of lymphocytic or collagenous gastroenteropathies.
Individual risk stratification of gastric cancer: evolving concepts and their impact on clinical practice.
Gastric cancer (GC) is the third leading cause of cancer-related death worldwide and it mostly develops in long-standing inflammatory conditions, and Helicobacter pylori-gastritis, in particular. Despite the increasing understanding of both the phenotypic alterations and the molecular mechanisms occurring during GC multi-step carcinogenesis, no reliable biomarker is available to be reliably implemented into GC secondary prevention strategies. Multidisciplinary diagnostic approaches integrating endoscopy, serology, histology and molecular profiling currently appears as the most appropriate approach for patients' stratification into different GC risk classes.
Pharmacological and alimentary alteration of the gastric barrier.
The gastric barrier contains several lines of defence which protect the epithelium from harmful microbes and toxins. Pre-mucosal defence mechanisms include secreted acid (HCl 0.1 mmol/L) and pepsin, which are capable of denaturing tissue. A tightly adherent mucous layer provides the next line of defence, and physically separates any potentially hazardous substance in the lumen from the mucosal surface. Apical secretion of HCO3(-) maintains a non-acidic microenvironment at the mucosal surface. Membrane-bound phospholipids repel soluble toxins, and sulphydryls scavenge reactive oxygen species. However, when noxious agents overwhelm these mechanisms, the epithelium is damaged. Herein, we discuss the pathological and physiological basis for several disease states which are associated with a breakdown in one or more components of the gastric barrier, including: Helicobacter pylori-associated gastritis, atrophic gastritis, stress-related mucosal disease, age-related gastropathy and portal hypertensive gastropathy. The effect of non-steroidal anti-inflammatory drugs and proton pump inhibitors on the gastric mucosa, is explored. Finally, we outline the alterations in mucosal defence caused by alcohol, caffeine, minerals and vitamins.
The stomach-brain axis.
The stomach has distinct functions in relation to the ingestion and handling of solids and liquids. These functions include storage of the food before it is gradually emptied into the duodenum, mechanical crushing of larger food particles to increase the surface area, secretion of an acidic enzyme rich gastric juice and mixing the ingested food with the gastric juice. In addition, the stomach 'senses' the composition of the gastric content and this information is passed via the vagal nerve to the lateral hypothalamus and the limbic system, most likely as palatability signals that influence eating behaviour. Other sensory qualities related to the stimulation of gastric tension receptors are satiety and fullness. Receptors that respond to macronutrient content or gastric wall tension influence appetite and meal related hormone responses. The ingestion of food - in contrast to an infusion of nutrients into the stomach - has distinct effects on the activation of specific brain regions. Brain areas such as thalamus, amygdala, putamen and praecuneus are activated by the ingestion of food. Gastric nutrient infusion evokes greater activation in the hippocampus and anterior cingulate. The brain integrates these interrelated neural and hormonal signals arising from the stomach as well as visual, olfactory and anticipatory stimuli that ultimately influence eating and other behavioural patterns. Furthermore, there is now good evidence from experimental studies that gastric afferents influence mood, and animal studies point towards the possibility that gastric dysfunction may be a risk factor for mood disorders such as anxiety and depression. The stomach is also not only colonised by Helicobacter pylori but a large array of bacteria. While there is sufficient evidence to suggest that H. pylori may alter caloric intake and mood, the role of other gastric microbiome for the brain function is unknown. To address this appropriate targeted gastric microbiome studies would be required instead of widely utilised opportunistic stool microbiome studies. In summary, it is now well established that there are important links between the brain and the stomach that have significant effects on gastric function. However, the stomach also influences the brain. Disturbances in the crosstalk between the stomach and the brain may manifest as functional GI disorders while disturbances in the stomach-brain communication may also result in an altered regulation of satiety and as a consequence may affect eating behaviour and mood. These observations may enable the identification of novel therapies targeted at the gastroduodenum that positively alter brain function and treat or prevent conditions such as obesity or functional gastrointestinal disorders.
The gastrointestinal microbiome - functional interference between stomach and intestine.
The gastrointestinal (GI) tract is a complex and dynamic network with interplay between various gut mucosal cells and their defence molecules, the immune system, food particles, and the resident microbiota. This ecosystem acts as a functional unit organized as a semipermeable multi-layer system that allows the absorption of nutrients and macromolecules required for human metabolic processes and, on the other hand, protects the individual from potentially invasive microorganisms. Commensal microbiota and the host are a unique entity in a continuum along the GI tract, every change in one of these players is able to modify the whole homeostasis. In the stomach, Helicobacter pylori is a gram-negative pathogen that is widespread all over the world, infecting more than 50% of the world's population. In this scenario, H. pylori infection is associated with changes in the gastric microenvironment, which in turn affects the gastric microbiota composition, but also might trigger large intestinal microbiota changes. It is able to influence all the vital pathways of human system and also to influence microbiota composition along the GI tract. This can cause a change in the normal functions exerted by intestinal commensal microorganisms leading to a new gastrointestinal physiological balance. This review focuses and speculates on the possible interactions between gastric microorganisms and intestinal microbiota and on the consequences of this interplay in modulating gut health.
The fight against gastric cancer - the IARC Working Group report.
Gastric cancer is the third cause of cancer death worldwide, and Helicobacter pylori infection causes almost 90% of non-cardia cancers, the predominant type. H. pylori infection is treatable, and in clinical trials there is evidence of a 30-40% reduction in incidence of gastric cancer among treated subjects. However, with a few exceptions, there are no public health programmes for gastric cancer prevention. In December 2013, the International Agency for Research on Cancer (IARC), organized a Working Group of international experts to discuss and make recommendations for gastric cancer control. The Working Group considered that the enormous burden of disease, which is not expected to decline in the coming decades, requires decisive public health action to include gastric cancer in cancer control programmes. Interventions should be tailored to the local conditions and consider population-based screening and eradication of H. pylori, in the context of evaluation of feasibility, efficacy and adverse consequences.
S3-guideline "helicobacter pylori and gastroduodenal ulcer disease" of the German society for digestive and metabolic diseases (DGVS) in cooperation with the German society for hygiene and microbiology, society for pediatric gastroenterology and nutrition e. V., German society for rheumatology, AWMF-registration-no. 021 / 001.
This guideline updates a prior consensus recommendation of the German Society for Digestive and Metabolic Diseases (DGVS) from 1996. It was developed by an interdisciplinary cooperation with representatives of the German Society for Hygiene and Microbiology, the Society for Pediatric Gastroenterology and Nutrition (GPGE), and the German Society for Rheumatology. The guideline is methodologically based on recommendations of the Association of the Scientific Medical Societies in Germany (AWMF) for providing a systematic evidence-based S 3 level consensus guideline and has also implemented grading criteria according to the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) process. Clinical applicability of study results as well as specifics for Germany in terms of epidemiology, antibiotic resistance status, diagnostics, and therapy were taken into account.
Different antibiotic susceptibility between antrum and corpus of the stomach, a possible reason for treatment failure of Helicobacter pylori infection.
AimTo assess whether antibiotic resistance varies between the antrum and corpus of the stomach of patients that are either Helicobacter pylori (H. pylori) therapy-naive or pre-treated.MethodsH. pylori strains were isolated from antrum and corpus biopsies from 66 patients that received a diagnostic gastroduodenoscopy for variant clinical indications. Antimicrobial susceptibility to amoxicillin, clarithromycin, tetracycline, metronidazole, levofloxacin and rifabutin was tested with the E-test method on Iso-Sensitest agar with 10 vol% defibrinated horse blood. In patients with a different antibiotic susceptibility pattern between the isolates from the antrum and corpus, DNA fingerprinting via random amplified polymorphic DNA analysis was performed to detect differences among DNA patterns of H. pylori isolates.ResultsPrimary, secondary and tertiary resistance to clarithromycin was 6.9%, 53.8% and 83.3%, retrospectively. Metronidazole and levofloxacin resistance also increased according to the number of previous treatments (17.2%, 69.2%, 83.3%; 13.8%, 23.1%, 33.3%). Tertiary resistance to rifabutin was detected in 12.5% of patients. In none of the 66 patients a resistance against amoxicillin or tetracycline was detectable. Discordant antibiotic susceptibility between antrum and corpus isolates for different antibiotics was seen in 15.2% (10/66) of the patients. Two out of those ten patients were naive to any H. pylori antibiotic treatment. The remaining eight patients previously received at least one eradication therapy. DNA fingerprinting analysis revealed no substantial differences among DNA patterns between antrum and corpus isolates in the majority of patients suggesting an infection with a single H. pylori strain.ConclusionDifferent antibiotic susceptibility between antrum and corpus biopsies is a common phenomenon and a possible explanation for treatment failure. Resistant H. pylori strains may be missed if just one biopsy from one anatomic site of the stomach is taken for H. pylori susceptibility testing.