The causal associations of circulating lipids with Barrett's Esophagus and Esophageal Cancer: a bi-directional, two sample mendelian randomization analysis.

OBJECTIVE: The causal associations of circulating lipids with Barrett's Esophagus (BE) and Esophageal Cancer (EC) has been a topic of debate. This study sought to elucidate the causality between circulating lipids and the risk of BE and EC. METHODS: We conducted two-sample Mendelian randomization (MR) analyses using single nucleotide polymorphisms (SNPs) of circulating lipids (n = 94,595 - 431,167 individuals), BE (218,792 individuals), and EC (190,190 individuals) obtained from the publicly available IEU OpenGWAS database. The robustness and reliability of the results were ensured by employing inverse-variance weighted (IVW), weighted median, MR-Egger, and MR-PRESSO methods. The presence of horizontal pleiotropy, heterogeneities, and stability of instrumental variables were assessed through MR-Egger intercept test, Cochran's Q test, and leave-one-out sensitivity analysis. Additionally, bidirectional MR and multivariable MR (MVMR) were performed to explore reverse causality and adjust for known confounders, respectively. RESULTS: None of the testing methods revealed statistically significant horizontal pleiotropy, directional pleiotropy, or heterogeneity. Univariate MR analyses using IVW indicated a robust causal relationship between increased triglycerides and BE (odds ratio [OR] = 1.79, p-value = 0.009), while no significant association with EC was observed. Inverse MR analysis indicated no evidence of reverse causality in the aforementioned outcomes. In MVMR analyses, elevated triglycerides (TRG) were significantly and positively associated with BE risk (OR = 1.79, p-value = 0.041). CONCLUSION: This MR study suggested that genetically increased triglycerides were closely related to an elevated risk of BE, potentially serving as a biomarker for the diagnosis of BE in the future.

miR-378a-5p represses Barrett's esophagus cells proliferation, migration and invasion through targeting TSPAN8.

Barrett's esophagus (BE) belongs to a pathological phenomenon occurring in the esophagus, this paper intended to unveil the underlying function of miR-378a-5p and its target TSPAN8 in BE progression. GEO analysis was conducted to determine differentially expressed genes in BE samples. Non-dysplastic metaplasia BE samples, high-grade dysplastic BE samples and controls were collected from subjects. CP-A and CP-B cells were exposed to bile acids (BA) to mimic gastroesophageal reflux in BE cells. RT-qPCR as well as western blot were applied for verifying expressions of miR-378a-5p, TSPAN8, CDX2 and SOX9. CCK-8, wound scratch together with Transwell assays were exploited for ascertaining cell proliferation, migration as well as invasion. The targeted relationship of miR-378a-5p and TSPAN8 could be verified by correlation analysis, dual-luciferase reporter experiment, and rescue experiments. Through analyzing GSE26886 dataset, we screened the most abundantly expressed gene TSPAN8 in BE samples. miR-378a-5p was reduced whereas TSPAN8 was elevated in CP-A as well as CP-B cells after triggering with BA. Knocking down TSPAN8 could counteract BA-triggered enhancement in BE cell proliferation, migration along with invasion. miR-378a-5p could suppress BE cell proliferation, and migration along with invasion via targeting TSPAN8. In BE, miR-378a-5p targeted TSPAN8 to inhibit BE cell proliferation, and migration along invasion. miR-378a-5p deletion or elevation of TSPAN8 may be key point in regulating CDX2 and SOX9 levels, thereby promoting BE formation.

Causal relationships between dietary habits and Barrett's esophagus risk: a univariable and multivariable Mendelian randomization study.

Aims: Dietary habits are reported to be associated with Barrett's esophagus (BE) risk; however, whether there is a causal relationship remains controversial. Here, we systematically examined the causal effects of genetically predicted dietary habits on BE risk through a Mendelian randomization (MR) analysis approach. Methods: Data for exposures were obtained from the UK Biobank (UKB), while the summary-level data for outcomes were obtained from a large sample-size GWAS meta-analysis. Genetic variants associated with 17 ordinary dietary habits at the genome-wide significance level were regarded as instrumental variables (IVs). Univariable and multivariable MR analyses were conducted to explore the causal relationships between dietary habits and BE risk. Sensitivity analyses were implemented to evaluate robustness of the results and determine the potential pleiotropy bias. Results: Univariable MR (UVMR) analysis showed that genetic predisposition to alcohol intake frequency, cooked vegetable intake, beef intake, bread intake, fresh fruit intake, salad/raw vegetable intake, and dried fruit intake were associated with BE risk, with all P values <0.05. After adjusting confounders, the effects of four dietary habits on BE risk persisted; multivariable MR (MVMR) analysis revealed that alcohol intake frequency (adjusted odds ratio (OR) = 1.74 (1.34, 2.27); P = 3.42 × 10-5) was causally associated with higher BE risk, the cooked vegetable intake (adjusted OR = 2.64 (1.16, 5.97); P = 0.02) had suggestively increased BE risk, while higher consumption of bread (adjusted OR = 0.54 (0.32-0.91); P = 0.02) and fresh fruit (adjusted OR = 0.34 (0.15, 0.77); P = 0.01) were suggestively associated with lower BE risk. Conclusions: These MR analyses demonstrate evidence of causal relationships between dietary habits and BE risk. These findings provide new insights into targeted dietary intervention strategies for BE prevention.

PARP1-targeted fluorescence molecular endoscopy as novel tool for early detection of esophageal dysplasia and adenocarcinoma.

BACKGROUND: Esophageal cancer is one of the 10 most common cancers worldwide and its incidence is dramatically increasing. Despite some improvements, the current surveillance protocol with white light endoscopy and random untargeted biopsies collection (Seattle protocol) fails to diagnose dysplastic and cancerous lesions in up to 50% of patients. Therefore, new endoscopic imaging technologies in combination with tumor-specific molecular probes are needed to improve early detection. Herein, we investigated the use of the fluorescent Poly (ADP-ribose) Polymerase 1 (PARP1)-inhibitor PARPi-FL for early detection of dysplastic lesions in patient-derived organoids and transgenic mouse models, which closely mimic the transformation from non-malignant Barrett's Esophagus (BE) to invasive esophageal adenocarcinoma (EAC). METHODS: We determined PARP1 expression via immunohistochemistry (IHC) in human biospecimens and mouse tissues. We also assessed PARPi-FL uptake in patient- and mouse-derived organoids. Following intravenous injection of 75 nmol PARPi-FL/mouse in L2-IL1B (n = 4) and L2-IL1B/IL8Tg mice (n = 12), we conducted fluorescence molecular endoscopy (FME) and/or imaged whole excised stomachs to assess PARPi-FL accumulation in dysplastic lesions. L2-IL1B/IL8Tg mice (n = 3) and wild-type (WT) mice (n = 2) without PARPi-FL injection served as controls. The imaging results were validated by confocal microscopy and IHC of excised tissues. RESULTS: IHC on patient and murine tissue revealed similar patterns of increasing PARP1 expression in presence of dysplasia and cancer. In human and murine organoids, PARPi-FL localized to PARP1-expressing epithelial cell nuclei after 10 min of incubation. Injection of PARPi-FL in transgenic mouse models of BE resulted in the successful detection of lesions via FME, with a mean target-to-background ratio > 2 independently from the disease stage. The localization of PARPi-FL in the lesions was confirmed by imaging of the excised stomachs and confocal microscopy. Without PARPi-FL injection, identification of lesions via FME in transgenic mice was not possible. CONCLUSION: PARPi-FL imaging is a promising approach for clinically needed improved detection of dysplastic and malignant EAC lesions in patients with BE. Since PARPi-FL is currently evaluated in a phase 2 clinical trial for oral cancer detection after topical application, clinical translation for early detection of dysplasia and EAC in BE patients via FME screening appears feasible.

CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett's-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions.

BACKGROUND & AIMS: Barrett's esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% of patients with Barrett's esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development. METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression targeting SCJ LGR5+ cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRASG12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). CONCLUSIONS: p16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

miRNA profiling of esophageal adenocarcinoma using transcriptome analysis.

Esophageal adenocarcinoma (EAC) occurs following a series of histological changes through epithelial-mesenchymal transition (EMT). A variable expression of normal and aberrant genes in the tissue can contribute to the development of EAC through the activation or inhibition of critical molecular signaling pathways. Gene expression is regulated by various regulatory factors, including transcription factors and microRNAs (miRs). The exact profile of miRs associated with the pathogenesis of EAC is largely unknown, though some candidate miRNAs have been reported in the literature. To identify the unique miR profile associated with EAC, we compared normal esophageal tissue to EAC tissue using bulk RNA sequencing. RNA sequence data was verified using qPCR of 18 selected genes. Fourteen were confirmed as being upregulated, which include CDH11, PCOLCE, SULF1, GJA4, LUM, CDH6, GNA12, F2RL2, CTSZ, TYROBP, and KDELR3 as well as the downregulation of UGT1A1. We then conducted Ingenuity Pathway Analysis (IPA) to analyze for novel miR-gene relationships through Causal Network Analysis and Upstream Regulator Analysis. We identified 46 miRs that were aberrantly expressed in EAC compared to control tissues. In EAC tissues, seven miRs were associated with activated networks, while 39 miRs were associated with inhibited networks. The miR-gene relationships identified provide novel insights into potentially oncogenic molecular pathways and genes associated with carcinogenesis in esophageal tissue. Our results revealed a distinct miR profile associated with dysregulated genes. The miRs and genes identified in this study may be used in the future as biomarkers and serve as potential therapeutic targets in EAC.

Accurate integration of single-cell DNA and RNA for analyzing intratumor heterogeneity using MaCroDNA.

Cancers develop and progress as mutations accumulate, and with the advent of single-cell DNA and RNA sequencing, researchers can observe these mutations and their transcriptomic effects and predict proteomic changes with remarkable temporal and spatial precision. However, to connect genomic mutations with their transcriptomic and proteomic consequences, cells with either only DNA data or only RNA data must be mapped to a common domain. For this purpose, we present MaCroDNA, a method that uses maximum weighted bipartite matching of per-gene read counts from single-cell DNA and RNA-seq data. Using ground truth information from colorectal cancer data, we demonstrate the advantage of MaCroDNA over existing methods in accuracy and speed. Exemplifying the utility of single-cell data integration in cancer research, we suggest, based on results derived using MaCroDNA, that genomic mutations of large effect size increasingly contribute to differential expression between cells as Barrett's esophagus progresses to esophageal cancer, reaffirming the findings of the previous studies.

The function and immune role of cuproptosis associated hub gene in Barrett's esophagus and esophageal adenocarcinoma.

Barrett's esophagus (BE) is a precancerous lesion of esophageal adenocarcinoma (EAC), with approximately 3-5% of patients developing EAC. Cuproptosis is a kind of programmed cell death phenomenon discovered in recent years, which is related to the occurrence and development of many diseases. However, its role in BE and EAC is not fully understood. We used single sample Gene Set Enrichment Analysis (ssGSEA) for differential analysis of BE in the database, followed by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and GSEA, Protein-Protein Interaction (PPI), Weighted Gene Co-expression Network Analysis (WGCNA), Receiver Operating Characteristic Curve (ROC) and finally Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry (IHC) of clinical tissues. Two hub genes can be obtained by intersection of the results obtained from the cuproptosis signal analysis based on BE. The ROC curves of these two genes predicted EAC, and the Area Under the Curve (AUC) values could reach 0.950 and 0.946, respectively. The mRNA and protein levels of Centrosome associated protein E (CENPE) and Shc SH2 domain binding protein 1 (SHCBP1) were significantly increased in clinical EAC tissues. When they were grouped by protein expression levels, high expression of CENPE or SHCBP1 had a poor prognosis. The CENPE and SHCBP1 associated with cuproptosis may be a factor promoting the development of BE into EAC which associated with the regulation of NK cells and T cells.

Genomic signatures of past and present chromosomal instability in Barrett's esophagus and early esophageal adenocarcinoma.

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

ESOMIR: a curated database of biomarker genes and miRNAs associated with esophageal cancer.

'Esophageal cancer' (EC) is a highly aggressive and deadly complex disease. It comprises two types, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), with Barrett's esophagus (BE) being the only known precursor. Recent research has revealed that microRNAs (miRNAs) play a crucial role in the development, prognosis and treatment of EC and are involved in various human diseases. Biological databases have become essential for cancer research as they provide information on genes, proteins, pathways and their interactions. These databases collect, store and manage large amounts of molecular data, which can be used to identify patterns, predict outcomes and generate hypotheses. However, no comprehensive database exists for EC and miRNA relationships. To address this gap, we developed a dynamic database named 'ESOMIR (miRNA in esophageal cancer) (https://esomir.dqweilab-sjtu.com)', which includes information about targeted genes and miRNAs associated with EC. The database uses analysis and prediction methods, including experimentally endorsed miRNA(s) information. ESOMIR is a user-friendly interface that allows easy access to EC-associated data by searching for miRNAs, target genes, sequences, chromosomal positions and associated signaling pathways. The search modules are designed to provide specific data access to users based on their requirements. Additionally, the database provides information about network interactions, signaling pathways and region information of chromosomes associated with the 3'untranslated region (3'UTR) or 5'UTR and exon sites. Users can also access energy levels of specific miRNAs with targeted genes. A fuzzy term search is included in each module to enhance the ease of use for researchers. ESOMIR can be a valuable tool for researchers and clinicians to gain insight into EC, including identifying biomarkers and treatments for this aggressive tumor. Database URL https://esomir.dqweilab-sjtu.com.

Ectopic expression of HNF4α in Het1A cells induces an invasive phenotype.

Barrett's oesophagus (BO) is a pathological condition in which the squamous epithelium of the distal oesophagus is replaced by an intestinal-like columnar epithelium originating from the gastric cardia. Several somatic mutations contribute to the intestinal-like metaplasia. Once these have occurred in a single cell, it will be unable to expand further unless the altered cell can colonise the surrounding squamous epithelium of the oesophagus. The mechanisms by which this happens are still unknown. Here we have established an in vitro system for examining the competitive behaviour of two epithelia. We find that when an oesophageal epithelium model (Het1A cells) is confronted by an intestinal epithelium model (Caco-2 cells), the intestinal cells expand into the oesophageal domain. In this case the boundary involves overgrowth by the Caco-2 cells and the formation of isolated colonies. Two key transcription factors, normally involved in intestinal development, HNF4α and CDX2, are both expressed in BO. We examined the competitive ability of Het1A cells stably expressing HNF4α or CDX2 and placed in confrontation with unmodified Het1A cells. The key result is that stable expression of HNF4α, but not CDX2, increased the ability of the cells to migrate and push into the unmodified Het1A domain. In this situation the boundary between the cell types is a sharp one, as is normally seen in BO. The experiments were conducted using a variety of extracellular substrates, which all tended to increase the cell migration compared to uncoated plastic. These data provide evidence that HNF4α expression could have a potential role in the competitive spread of BO into the oesophagus as HNF4α increases the ability of cells to invade into the adjacent stratified squamous epithelium, thus enabling a single mutant cell eventually to generate a macroscopic patch of metaplasia.

Microarchitectures of Barrett's esophagus associated with DNA methylation status.

Aim: DNA methylation is involved in esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE). Microarchitectures of on-neoplastic BE associated with DNA methylation status were examined using magnifying narrow-band imaging (NBI) endoscopy. Patients and methods: Using biopsies from non-neoplastic BE without cancer (n = 66; N group), with EAC (n = 27; ADJ group) and EAC tissue (n = 22; T group), methylation of N33, DPYS, SLC16A12, miR124a3 and miR34bc genes were quantified. Magnifying NBI features of non-neoplastic BE were classified according to their morphologies. Results: The ADJ and T groups presented higher DNA methylation compared with the N group. Magnifying NBI endoscopic features of non-neoplastic BE also correlated with DNA methylation as an independent factor. Conclusion: Microarchitectures of BE visualized by magnifying NBI endoscopy correlated with DNA methylation.

Causal relationship between gastroesophageal reflux disease, Barrett's esophagus, and epilepsy: A bidirectional Mendelian randomization study.

BACKGROUND: The incidence of gastroesophageal reflux disease (GERD) has been shown to be elevated in individuals with epilepsy. Traditional observational studies have led to a limited understanding of the effects of GERD and BE on epilepsy due to the interference of reverse causation and potential confounders. METHODS: We conducted a bidirectional two-sample Mendelian randomization (MR) analysis to determine whether GERD and BE can increase the risk of epilepsy. Genome-wide association study data on epilepsy and its subgroups were obtained from the International League Against Epilepsy consortium for primary analysis using three MR approaches and the FinnGen consortium for replication and meta-analysis. We calculated causal estimates between the two esophageal diseases and epilepsy using the inverse-variance weighted method. Sensitivity analysis was conducted to detect heterogeneity and pleiotropy. RESULTS: We found a potential effect of genetically predicted GERD on the risk of epilepsy (odds ratio [OR] = 1.078; 95% confidence interval [CI], 1.014-1.146, p = .016). Specifically, GERD showed an effect on the risk of generalized epilepsy (OR = 1.163; 95% CI, 1.048-1.290, p = .004) but not focal epilepsy (OR = 1.059, 95% CI, 0.992-1.131, p = .084). Notably, BE did not show a significant causal relationship with the risks of generalized and focal epilepsy. CONCLUSIONS: Under MR assumptions, our findings suggest a potential risk-increasing effect of GERD on epilepsy, especially generalized epilepsy. Considering the exploratory nature of our study, the association between GERD and epilepsy needs to be confirmed by future prospective studies.

MicroRNA dysregulation and therapeutic opportunities in esophageal diseases.

MicroRNAs (miRNAs) are a class of small endogenous RNA molecules between 18 and 25 nucleotides long. The primary function of miRNAs is in the posttranscriptional regulation of mRNA targets through RNA interference culminating in mRNA degradation or translational repression. MiRNAs are fundamental in physiological and pathological processes such as cell proliferation, differentiation, apoptosis, and inflammation. Among this includes the uncovered potential of miRNAs in overall esophageal disease with a focus on the clinicopathologic allergic disease eosinophilic esophagitis (EoE), gastroesophageal reflux disease (GERD), and the tumorigenic continuum from Barrett's esophagus (BE) toward esophageal adenocarcinoma (EAC). Although these pathologies are distinct from one another, they share pathophysiological elements such as an intense inflammatory milieu, esophageal dysfunction, and as presented in this review, an overlap in miRNA expression which contributes to overall esophageal disease. The overlap in the dysregulated miRNA transcriptome of these pathologies highlights the key role miRNAs play in contributing to esophageal disease progression. Owing to this notable dysregulation, there is an attractive utility for miRNAs as diagnostic and prognostic biomarkers in esophageal diseases that already require invasive endoscopies and biopsy retrieval. In this review miRNAs within EoE, GERD, BE, EAC, and esophageal achalasia are discussed, as well as reviewing a core set of miRNAs shared in the disease progression among some of these pathologies, along with the potential utility of targeting miRNAs as therapeutic options in overall esophageal disease.

Dissecting the genetic heterogeneity of gastric cancer.

BACKGROUND: Gastric cancer (GC) is clinically heterogenous according to location (cardia/non-cardia) and histopathology (diffuse/intestinal). We aimed to characterize the genetic risk architecture of GC according to its subtypes. Another aim was to examine whether cardia GC and oesophageal adenocarcinoma (OAC) and its precursor lesion Barrett's oesophagus (BO), which are all located at the gastro-oesophageal junction (GOJ), share polygenic risk architecture. METHODS: We did a meta-analysis of ten European genome-wide association studies (GWAS) of GC and its subtypes. All patients had a histopathologically confirmed diagnosis of gastric adenocarcinoma. For the identification of risk genes among GWAS loci we did a transcriptome-wide association study (TWAS) and expression quantitative trait locus (eQTL) study from gastric corpus and antrum mucosa. To test whether cardia GC and OAC/BO share genetic aetiology we also used a European GWAS sample with OAC/BO. FINDINGS: Our GWAS consisting of 5816 patients and 10,999 controls highlights the genetic heterogeneity of GC according to its subtypes. We newly identified two and replicated five GC risk loci, all of them with subtype-specific association. The gastric transcriptome data consisting of 361 corpus and 342 antrum mucosa samples revealed that an upregulated expression of MUC1, ANKRD50, PTGER4, and PSCA are plausible GC-pathomechanisms at four GWAS loci. At another risk locus, we found that the blood-group 0 exerts protective effects for non-cardia and diffuse GC, while blood-group A increases risk for both GC subtypes. Furthermore, our GWAS on cardia GC and OAC/BO (10,279 patients, 16,527 controls) showed that both cancer entities share genetic aetiology at the polygenic level and identified two new risk loci on the single-marker level. INTERPRETATION: Our findings show that the pathophysiology of GC is genetically heterogenous according to location and histopathology. Moreover, our findings point to common molecular mechanisms underlying cardia GC and OAC/BO. FUNDING: German Research Foundation (DFG).

Comprehensive DNA methylation profiling of Barrett's esophagus and esophageal adenocarcinoma in Japanese patients.

Molecular mechanisms of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remain unclear in Japanese patients. Japanese EACs frequently have underlying short length BE: short-segment BE (SSBE), for which, neoplastic potential remains unclear. We performed comprehensive methylation profiling of EAC and BE in Japanese patients, mostly comprised with SSBE. Using three different groups of biopsies obtained from non-neoplastic BE from patients without cancer (n = 50; N group), with EAC (n = 27; ADJ group) and EAC (n = 22; T group), methylation statuses of nine candidate genes (N33, DPYS, SLC16A12, CDH13, IGF2, MLF1, MYOD1, PRDM5, and P2RX7) were examined by the bisulfite pyrosequencing. Reduced representation bisulfite sequencing was performed to characterize the genome-wide methylation status in 32 samples (12 from N, 12 ADJ, and 8 from T groups). In the candidate approach, methylation levels of N33, DPYS, and SLC16A12 were higher in ADJ and T groups compared to that in N group. The ADJ group was an independent factor for higher DNA methylation in non-neoplastic BE. The genome-wide approach demonstrated an increase of hypermethylation from ADJ to T groups relative to N group near the transcription start sites. Among gene groups hypermethylated in ADJ and T groups (n = 645) and T group alone (n = 1438), 1/4 and 1/3 were overlapped with downregulated genes in the microarray data set, respectively. Accelerated DNA methylation is observed in EAC and underlying BE in Japanese patients, mostly comprised with SSBE, highlighting the potential impact of methylation in early carcinogenesis.

A Method for Increasing the Robustness of Stable Feature Selection for Biomarker Discovery in Molecular Medicine Developed Using Serum Small Extracellular Vesicle Associated miRNAs and the Barrett's Oesophagus Disease Spectrum.

The biomarker development field within molecular medicine remains limited by the methods that are available for building predictive models. We developed an efficient method for conservatively estimating confidence intervals for the cross validation-derived prediction errors of biomarker models. This new method was investigated for its ability to improve the capacity of our previously developed method, StaVarSel, for selecting stable biomarkers. Compared with the standard cross validation method, StaVarSel markedly improved the estimated generalisable predictive capacity of serum miRNA biomarkers for the detection of disease states that are at increased risk of progressing to oesophageal adenocarcinoma. The incorporation of our new method for conservatively estimating confidence intervals into StaVarSel resulted in the selection of less complex models with increased stability and improved or similar predictive capacities. The methods developed in this study have the potential to improve progress from biomarker discovery to biomarker driven translational research.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Association of educational attainment with esophageal cancer, Barrett's esophagus, and gastroesophageal reflux disease, and the mediating role of modifiable risk factors: A Mendelian randomization study.

Background: Observational studies have reported that educational attainment has been related to the risk of esophageal cancer (EC) and its precancerous lesions. However, the causal relationship remains controversial. We aimed to apply the Mendelian randomization (MR) design to determine the causal associations between genetically predicted educational attainment and EC, Barrett's esophagus (BE), and gastroesophageal reflux disease (GERD), and to explore whether modifiable risk factors play a mediating role. Methods: Using summary statistics from genome-wide association studies (GWASs) based on European ancestry individuals of several years in education (EduYears, primary analysis, n = 293,723), college completion (College, secondary analysis, n = 95,427), EC (n = 420,531), BE (n = 361,194), and GERD (n = 420,531), genetic associations between two education phenotypes and EC, BE, and GERD were tested by two-sample MR analyses. Then, two-step MR mediation analyses were used to assess the proportion of the aforementioned association that might be mediated by body mass index (BMI), major depressive disorder (MDD), smoking, drinking, carbohydrates, fat, and protein intake. Results: Genetically predicted EduYears was negatively associated with the risk of EC, BE, and GERD {odds ratio (OR), 0.64 [95% confidence interval (CI) 0.44-0.94], 0.86 (95% CI, 0.75-0.99), and 0.62 (95%CI, 0.58-0.67)}. EduYears was negatively associated with BMI, MDD, and smoking (range of OR: 0.76-0.84). There were positive associations between BMI, smoking with EC, BE, and GERD, as well as between MDD with GERD (range of OR: 1.08-1.50). For individual mediating effect, BMI and smoking mediated 15.75 and 14.15% of the EduYears-EC association and 15.46 and 16.85% of the EduYears-BE association. BMI, MDD, and smoking mediated 5.23, 4.98, and 4.49% of the EduYears-GERD association. For combined mediation, the aforementioned mediators explained 26.62, 28.38, and 11.48% of the effect of EduYears on EC, BE, and GERD. The mediating effects of drinking and dietary composition were not significant in the effect of education on EC, BE, and GERD. Conclusion: Our study supports that genetically predicted higher educational attainment has a protective effect on EC, BE, and GERD, and is partly mediated by reducing adiposity, smoking, and depression.