Subtypes of Barrett’s oesophagus and oesophageal adenocarcinoma based on genome-wide methylation analysis

ObjectiveTo identify and characterise DNA methylation subtypes in oesophageal adenocarcinoma (EAC) and its precursor Barrett’s oesophagus (BE).DesignWe performed genome-wide DNA methylation profiling on samples of non-dysplastic BE from cancer-free patients (n=59), EAC (n=23), normal squamous oesophagus (n=33) and normal fundus (n=9), and identified methylation subtypes using a recursively partitioned mixture model. We assessed genomic alterations for 9 BE and 22 EAC samples with massively parallel sequencing of 243 EAC-associated genes, and we conducted integrative analyses with transcriptome data to identify epigenetically repressed genes. We also carried out in vitro experiments treating EAC cell lines with 5-Aza-2'-Deoxycytidine (5-Aza-dC), short hairpin RNA knockdown and anticancer therapies.ResultsWe identified and validated four methylation subtypes of EAC and BE. The high methylator subtype (HM) of EAC had the greatest number of activating events in ERBB2 (p<0.05, Student’s t-test) and the highest global mutation load (p<0.05, Fisher’s exact test). PTPN13 was silenced by aberrant methylation in the HM subtype preferentially and in 57% of EACs overall. In EAC cell lines, 5-Aza-dC treatment restored PTPN13 expression and significantly decreased its promoter methylation in HM cell lines (p<0.05, Welch’s t-test). Inhibition of PTPN13 expression in the SK-GT-4 EAC cell line promoted proliferation, colony formation and migration, and increased phosphorylation in ERBB2/EGFR/Src kinase pathways. Finally, EAC cell lines showed subtype-specific responses to topotecan, SN-38 and palbociclib treatment.ConclusionsWe identified and characterised methylator subtypes in BE and EAC. We further demonstrated the biological and clinical relevance of EAC methylator subtypes, which may ultimately help guide clinical management of patients with EAC.

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Long-term proton pump induced hypergastrinaemia does induce lineage-specific restitution but not clonal expansion in benign Barrett's oesophagus in vivo

Gut ◽

10.1136/gut.2009.186775 ◽

2009 ◽

Vol 59 (2) ◽

pp. 156-163 ◽

Cited By ~ 18

Author(s):

Jolanta A Obszynska ◽

Paul A Atherfold ◽

Manoj Nanji ◽

Deborah Glancy ◽

Sonia Santander ◽

...

Keyword(s):

Proton Pump ◽

Biological Effects ◽

T Test ◽

Barrett’S Oesophagus ◽

Segment Length ◽

Term Therapy ◽

Barrett's Oesophagus ◽

Paired T Test

BackgroundBarrett's oesophagus is a common premalignant lesion caused partly by acid reflux. Although the requisite therapy, proton pump inhibitors (PPIs), have been implicated in the progression of Barrett's oesophagus in animal models, harmful effects of prolonged PPI therapy in Barrett's oesophagus is both inconclusive and controversial. We therefore aimed to test the role of PPI-induced hypergastrinaemia in vitro and see whether any biological parameters were useful surrogates of long-term therapy in man.MethodsWe undertook detailed serological and tissue assessment of gastrin and CCK2 receptors in 90 patients randomised to different doses of PPI therapy during a detailed 2-year follow-up. We also undertook a comprehensive study of cell models to study the consequential biological effects of gastrin on the mucosa.ResultsGastrin and its cognate receptor CCK2R were expressed highest in the stomach, then less in Barrett's oesophagus and least in squamous oesophagus (SqE) (n=20 paired t-test, p<0.01). Analysis of the change in Barrett's oesophagus segment length change in 70 patients who were randomised to high or low PPI dose showed no difference over 2 years (n=70 t-test, p=0.8). Prolonged PPI use did, however, increase the serum gastrin, (36 pg/ml±57 pg/ml to 103 pg/ml±94 pg/ml (paired t test, p<0.05)). In vitro gastrin also induced changes in OE33(E)cckr Barrett's oesophagus cells, but not OE21(E)cckr squamous cells, transfected with CCK2R; migration was induced by 1 ng/ml of gastrin but proliferation only increased with 100 ng/ml (paired t-test, p<0.01) and both were abolished by antagonists.ConclusionWhile the short-term effects of gastrin enhance epithelial restitution in Barrett's oesophagus (but not squamous mucosa) there is no clinical evidence that Barrett's oesophagus length expands over time. This study, which is the largest and longest term randomised controlled trial of gastrin biology in Barrett's oesophagus, is further proof of the clinical safety of PPI therapy.

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Genome-wide association studies in oesophageal adenocarcinoma and Barrett's oesophagus: a large-scale meta-analysis

The Lancet Oncology ◽

10.1016/s1470-2045(16)30240-6 ◽

2016 ◽

Vol 17 (10) ◽

pp. 1363-1373 ◽

Cited By ~ 67

Author(s):

Puya Gharahkhani ◽

Rebecca C Fitzgerald ◽

Thomas L Vaughan ◽

Claire Palles ◽

Ines Gockel ◽

...

Keyword(s):

Large Scale ◽

Association Studies ◽

Meta Analysis ◽

Genome Wide Association ◽

Barrett’S Oesophagus ◽

Oesophageal Adenocarcinoma ◽

Genome Wide Association Studies ◽

Barrett's Oesophagus ◽

Genome Wide

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Leptin activates Akt in oesophageal cancer cells via multiple atorvastatin-sensitive small GTPases

Molecular and Cellular Biochemistry ◽

10.1007/s11010-021-04067-8 ◽

2021 ◽

Author(s):

Ian L. P. Beales ◽

Olorunseun O. Ogunwobi

Keyword(s):

Oesophageal Cancer ◽

Small Gtpases ◽

Dominant Negative ◽

Kinase Assay ◽

Barrett’S Oesophagus ◽

Oesophageal Adenocarcinoma ◽

Barrett's Oesophagus ◽

Akt Activation ◽

Adenocarcinoma Cells

AbstractObesity is a risk factor for Barrett’s oesophagus and oesophageal adenocarcinoma. Adipose tissue secretes the hormone leptin. Leptin is a growth factor for several cell types, including Barrett’s cells and oesophageal adenocarcinoma cells. Statins are associated with reduced rates of Barrett’s oesophagus and oesophageal cancer and exhibit anti-cancer effects in vitro. The mechanisms of these effects are not fully established. We have examined the effects of leptin and the lipid-soluble statin, atorvastatin, on signalling via monomeric GTP-binding proteins and Akt. Proliferation and apoptosis were assessed in OE33 cells. Akt activity was quantified by cell-based ELISA and in vitro kinase assay. Specific small-molecule inhibitors and a dominant-negative construct were used to reduce Akt activity. Small GTPases were inhibited using transfection of dominant-negative plasmids, prenylation inhibitors and pretreatment with atorvastatin. Leptin stimulated Akt activity and cell proliferation and inhibited camptothecin-induced apoptosis in an Akt-sensitive manner. Leptin induced phosphorylation of Bad and FOXO1 in an Akt-sensitive manner. Leptin activated Ras, Rac, RhoA and cdc42. Transfection of dominant-negative plasmids confirmed that leptin-induced Akt activation required Ras, RhoA cdc42 but not Rac. Atorvastatin inhibited leptin-induced activation of Ras, RhoA, cdc42 and Akt. Co-treatment with mevalonate prevented these effects of atorvastatin. The protein kinase Akt is essential to the growth-promoting and anti-apoptotic effects of leptin in oesophageal adenocarcinoma cells. Akt is activated via Ras-, Rho- and cdc42-dependant pathways. Atorvastatin reduces leptin-induced Akt activation by inhibiting prenylation of small GTPases. This may explain the reduced incidence of oesophageal adenocarcinoma in statin-users.

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TMOD-05. GENOME-WIDE DNA METHYLATION PROFILE: A POWERFUL STRATEGY TO RECAPITULATE HETEROGENEITY OF PEDIATRIC BRAIN TUMORS IN PRIMARY CELL LINES

Neuro-Oncology ◽

10.1093/neuonc/noab090.146 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i36-i36

Author(s):

Lucia Pedace ◽

Simone Pizzi ◽

Maria Vinci ◽

Giulia Pericoli ◽

Giuseppina Catanzaro ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Pediatric Brain Tumors ◽

Genetic Alterations ◽

In Vitro Models ◽

Primary Cell ◽

Genome Wide ◽

Primary Cell Lines ◽

Pediatric Brain

Abstract Background Development of in vitro models of pediatric brain tumors (pBT) is instrumental for both understanding the contributing oncogenic molecular mechanisms and identifying and testing new therapeutic strategies. Primary cell lines should be established and managed to prevent epigenetic and genetic alterations and thus recapitulating the original tumor. DNA methylation (DM) is a stable epigenetic modification, altered in cancer and recently used to classify tumors. We aim to apply DM and Copy Number Variation (CNV) profiling to characterize pBT primary cell lines and tumors. Methods We investigated 34 pBT tissues from different histology paired to 52 their derived primary cultures in both 2D and 3D conditions, as stem-cells or in serum-supplemented medium, and both short and long-terms in culture. We studied 18 additional pBT-derived cell-lines, 9 organoids, 5 commercial cell-lines, and 122 pBT tissues from the same histological categories, as controls, for a total of 240 genome-wide DM profiles. We analyzed DM and CNV profiles by using Illumina EPIC-arrays. By means of a bump hunting strategy, we identified differentially methylated regions in faithful vs unfaithful cell lines, and performed a functional characterization using over-representation analysis. Results The 69% (25/36) of cells at early passages retained genetic alteration and the same DM patterns of the original tumors, with no differences related to 2D/3D methods or the presence of serum in media. The 70% (24/34) of primary cell lines analyzed at later passages (>5 or >14 days in culture) diverged from the primary tumor, the totality of those cultured with serum. All divergent cells clustered together acquiring common deregulated epigenetic signature induced by serum culture media, 2D methods and longer time in culture. Conclusions We have shown that global DM profiles, along with CNV analysis are useful tools to detect the recapitulation of pBT-derived primary cell-lines from the original tumor. Whatever subgroups tested, our results suggest that in vitro models should be passaged as little as possible to retain the epigenetic and genetic alterations of the tumors and thus to be considered relevant for basic and translational biology.

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In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer

Biomedicines ◽

10.3390/biomedicines8080233 ◽

2020 ◽

Vol 8 (8) ◽

pp. 233

Author(s):

Christina U. Köhler ◽

Michael Walter ◽

Kerstin Lang ◽

Sabine Plöttner ◽

Florian Roghmann ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Urothelial Cancer ◽

Area Under The Curve ◽

Region Of Interest ◽

Urine Samples ◽

Genome Wide ◽

In Vitro Data

We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this “bladder-specific” region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.

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P-OGC95 Understanding the molecular age of Barrett’s oesophagus in a population-representative sample of patients

British Journal of Surgery ◽

10.1093/bjs/znab430.222 ◽

2021 ◽

Vol 108 (Supplement_9) ◽

Author(s):

Talita Oliveira ◽

McKenna Lewis ◽

Laura Smyth ◽

Richard Turkington ◽

Amy Jayne McKnight ◽

...

Keyword(s):

Molecular Clock ◽

Population Based ◽

Barrett’S Oesophagus ◽

Oesophageal Adenocarcinoma ◽

Patient Specific ◽

Biological Aging ◽

Computational Tools ◽

Barrett's Oesophagus ◽

Genome Wide ◽

Aging Rates

Abstract Background The incidence of oesophageal adenocarcinoma (OAC) increases dramatically with patient age but only a small proportion of patients with diagnosed Barrett’s oesophagus (BO), the precursor to OAC, will develop dysplasia and/or cancer. Beyond chronological age, biomarkers of progression that capture biological aging offer largely untapped potential for objectively identifying BO patients at highest risk of progression, who could undergo personalised surveillance at shorter intervals. We have developed computational tools to determine tissue-specific aging using genome-wide methylation data as a “molecular clock” for estimating patient-specific BO dwell times at the time of incident diagnosis that cannot be clinically measured by other means. Methods Using the population-based Northern Ireland BO register in a retrospective study, we have identified 46 non-dysplastic BO patients who have 2-4 serial endoscopic biopsies each, and have not progressed to OAC (age range 29-77 years). FFPE biopsies for 10 age-matched patients who had prevalent HGD/OAC at index BO diagnosis were also retrieved. DNA has been extracted, quantified using fluorescence, quality checked through qPCR, and prepared for Illumina EPIC methylation arrays. We created a Python package called “MethylDrift” to determine genome-wide aging rates in patient data. Model outputs are used in the molecular clock for BO tissue age. Results We used MethylDrift to quantify aging rates in both cross-sectional data (population-level epigenetic drift) and longitudinal data within the same patients to obtain individual aging rates. Computational analyses using our previously developed Bayesian framework for the BO molecular clock will be applied to estimate the molecular age of BO in patients, i.e., how long the patient has been living with BO since onset of metaplasia. Results will be compared between age groups, birth cohorts, sex, and importantly between dysplastic and non-dysplastic BO to evaluate biomarker potential. Data analysis is ongoing, and the final results will be presented at the meeting. Conclusions Our results from this nested case-control study demonstrate feasibility and generate pilot data on molecular age as a proxy of BO duration at the time of incident diagnosis, in a large population-based registry of patients with BO. This will inform our computational tools for determining biological aging and can be applied in future work to investigate progression risk according to molecular age. Ultimately, this biomarker could inform surveillance frequency for BO patients, enable earlier detection of neoplastic progression, leading to improved patient outcomes and optimal distribution of limited endoscopy capacity for surveillance.

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New models of neoplastic progression in Barrett's oesophagus

Biochemical Society Transactions ◽

10.1042/bst0380331 ◽

2010 ◽

Vol 38 (2) ◽

pp. 331-336 ◽

Cited By ~ 8

Author(s):

Kirill Pavlov ◽

Carlo C. Maley

Keyword(s):

Tissue Culture ◽

Cell Lines ◽

Computational Models ◽

Evolutionary Dynamics ◽

Kinase Inhibitor ◽

Three Dimensional ◽

Barrett’S Oesophagus ◽

Oesophageal Adenocarcinoma ◽

Neoplastic Progression ◽

Barrett's Oesophagus

Research in Barrett's oesophagus, and neoplastic progression to OAC (oesophageal adenocarcinoma), is hobbled by the lack of good pre-clinical models that capture the evolutionary dynamics of Barrett's cell populations. Current models trade off tractability for realism. Computational models are perhaps the most tractable and can be used both to interpret data and to develop intuitions and hypotheses for neoplastic progression. Tissue culture models include squamous cell lines, Barrett's oesophagus cell lines and OAC cell lines, although it was recognized recently that BIC-1, SEG-1 and TE-7 are not true OAC cell lines. Some of the unrealistic aspects of the micro-environment in two-dimensional tissue culture may be overcome with the development of three-dimensional organotypic cultures of Barrett's oesophagus. The most realistic, but least tractable, model is a canine surgical model that generates reflux and leads to an intestinal metaplasia. Alternatively, rat surgical models have gained popularity and should be tested for the common genetic features of Barrett's oesophagus neoplastic progression in humans including loss of CDKN2A (cyclin-dependent kinase inhibitor 2A) and TP53 (tumour protein 53), generation of aneuploidy and realistic levels of genetic diversity. This last feature will be important for studying the effects of cancer-prevention interventions. In order to study the dynamics of progression and the effects of an experimental intervention, there is a need to follow animals longitudinally, with periodic endoscopic biopsies. This is now possible and represents an exciting opportunity for the future.

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THU0004 GENOME-WIDE DNA METHYLATION PROFILING IN MONOCYTES FROM PRIMARY ANTIPHOSPHOLIPID SYNDROME PATIENTS IDENTIFIES AN ABERRANT METHYLATION SIGNATURE ASSOCIATED WITH THEIR ATHEROTHROMBOTIC PHENOTYPE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5358 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 214.2-215

Author(s):

C. Perez-Sanchez ◽

A. M. Patiño-Trives ◽

M. A. Aguirre ◽

P. S. Laura ◽

M. Luque-Tévar ◽

...

Keyword(s):

Dna Methylation ◽

Association Studies ◽

Methylation Profile ◽

Aberrant Methylation ◽

Functional Classification ◽

Research Support ◽

Genome Wide

Background:Recent studies underlined the crucial role of DNA methylation in several autoimmune diseases by altering gene expression profiles, thus influencing disease severity. Yet, aberrant methylation patterns in monocytes, key players in the pathogenesis of APS patients, has not been evaluated.Objectives:To analyze the genome-wide DNA methylation profile of monocytes from APS patients and its relationship with the cardiovascular (CV) pathology. 2. To evaluate the role of antiphospholipid antibodies (aPL) in the regulation of this process.Methods:Thirty-three APS patients and 15 healthy donors (HD) were included in the study. Monocytes were isolated from peripheral blood by positive immunomagnetic selection. The Illumina Infinium Methylation EPIC Beadchip was used to obtain DNA methylation profiles across approximately 850,000 CpGs (TSS1500, TSS200, 5UTR, 3UTR, first exon, intergenic, gene body). Beta values (β) estimating methylation levels were obtained at each CpG site, and differentially methylated genes (DMG) between APS and HD were identified. Functional classification of that genes was carried out by gene ontology analysis (PANTHER database). Gene expression of selected DMG genes was evaluated by RT-PCR. CV-risk parameters, including carotid intima-media thickness (CIMT) and microvascular endothelial function were further assessed, and correlation/association studies were developed with clinical and analytical variables. The effects of aPLs were also evaluated byin vitrostudies.Results:Genome-wide DNA methylation analysis identified 813 DMG, including 279 hypomethylated and 534 hypermethylated. Functional classification of these genes revealed signatures associated with biological processes and pathways related to their clinical profile, including immune response, adhesion, oxidative stress and vascular signaling. Correlation and association studies showed that the methylation levels of genes related to immune response were associated with the CV-risk score, aGAPSS (CCR2, TXLNB, GLIPR), type of thrombosis (SIGLEC11, COLEC11, LRRC16A, AHSA1, TRIL) and aPL titers (CLEC4G, RGS4, HLA-DPA1, GBP6, RAET1E, HLA-G, HLA-DPA1, HLA-H, TXLNB). Besides, methylation levels of DMG related to vascular signaling and adhesion processes were associated with the presence of thrombotic recurrences (VEGFA, MAPK14, ITGA8, EPCAM, PCDHA6, DLG1) as well as with traditional CV-risk factor such as hypertension and dyslipidemia (ITGA11, DSCAM, CLEC4F, CDH4, LTBP2, PCDHB14). In addition, methylation levels of DMG genes related to oxidative stress (GP2, PGD, ADH1) were associated with microvascular endothelial dysfunction. An altered mRNA expression of some of those genes with aberrant methylation and related to increased CV-risk and thrombotic recurrences in APS was also identified. Both, abnormal methylation and transcription levels of several genes were further associated with a pathological increase of the CIMT. Finally, in vitro studies supported the role of aPLs as key players in the altered methylation and transcriptomic profiles of APS patients.Conclusion:APS patients showed an impaired methylation profile in monocytes of genes associated with clinical features of the disease, including aPL titers, CV risk, thrombotic recurrences, endothelial dysfunction and early atherosclerosis. These results offered a map to the monocytes methylome and shed light on the pathophysiology of APS, paving the way for the development of new, more effective biomarkers and therapeutics.Acknowledgments:Funded by ISCIII (PI18/0837 and RIER RD16/0012/0015) co-funded with FEDER.Disclosure of Interests:Carlos Perez-Sanchez: None declared, Alejandra M. Patiño-Trives: None declared, Maria A Aguirre: None declared, Pérez Sánchez Laura: None declared, María Luque-Tévar: None declared, Iván Arias de la Rosa: None declared, Carmen Torres-Granados: None declared, Maria del Carmen Abalos-Aguilera: None declared, Pedro Seguí Azpilcueta: None declared, Javier Rodríguez: None declared, Esteban Ballester: None declared, Nuria Barbarroja Puerto Grant/research support from: ROCHE and Pfizer., Speakers bureau: ROCHE and Celgene., Eduardo Collantes Estevez Grant/research support from: ROCHE and Pfizer, Speakers bureau: ROCHE, Lilly, Bristol and Celgene, Chary Lopez-Pedrera Grant/research support from: ROCHE and Pfizer.

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