Helicobacter pylori testing prior to or at gastric cancer diagnosis and survival in a diverse US patient population.

BACKGROUND: Gastric cancer (GC) accounts for the greatest disparity in cancer mortality between Black and White Americans. Although clinical trials have shown that Helicobacter pylori (Hp) treatment reduces risk of GC, Hp testing and treatment is not consistently performed in the US, and may offer an opportunity to improve survival. METHODS: In a diverse retrospective cohort of 99 GC cases diagnosed at Duke University from 2002-2020 (57% Black; 43% white), we examined the association of Hp testing and treatment prior to or at cancer diagnosis with overall survival using Cox regression analyses to calculate adjusted hazards ratios (HRs) and 95% confidence intervals (CIs). RESULTS: Overall, 62% of patients were tested for Hp prior to or at GC diagnosis. Of those, 25% tested positive and were treated < 1 year prior to or at diagnosis, 15% tested positive and were treated ≥ 1 year prior to diagnosis, 6% tested positive without evidence of treatment, and 54% tested negative. Compared to never tested, Hp testing and treatment < 1 year prior to or at diagnosis was associated with a significantly reduced likelihood of death (HR 0.21, 95% CI 0.08-0.58). The benefit of any Hp test and treat prior to or at GC diagnosis was significant even among stage IV patients only (HR, 0.22; 95% CI 0.05-0.96). CONCLUSIONS: These findings support Hp testing and treatment for patients at risk of or diagnosed with GC, and suggest Hp treatment may provide an opportunity to reduce GC mortality disparities in the US.

Type 2 Diabetes Mellitus and Helicobacter pylori Eradication in a Clinical Population.

OBJECTIVES: Eradication of Helicobacter pylori reduces the risk of gastric cancer (GC). Individuals with type 2 diabetes mellitus (T2DM) are known to be at increased risk for GC. In a cohort of H. pylori-positive individuals, we assessed whether those with T2DM were at risk of persistent infection following H. pylori treatment compared with individuals without T2DM. METHODS: A random subset of all individuals diagnosed as having H. pylori without intestinal metaplasia at endoscopy from 2015 to 2019 were stratified evenly by race (Black and White). After excluding those with T1DM and those without eradication testing after H. pylori treatment, logistic regression analysis was used to determine the association of T2DM with the risk of persistent H. pylori infection following treatment. RESULTS: In 138 patients, H. pylori eradication rates did not differ between the 27% of individuals with T2DM compared to those without (81.1% vs 81.2%). After adjusting for age, race, and insurance status, we found no significant increased risk of persistent H. pylori infection for individuals with T2DM (odds ratio 1.40; 95% confidence interval 0.49-3.99). CONCLUSIONS: H. pylori eradication rates do not differ by T2DM status, providing support for clinical trials of H. pylori eradication to reduce GC incidence among high-risk populations in the United States, such as individuals with T2DM.

The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett's Neoplasia.

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

Individualized ergonomic wellness approach for the practicing gastroenterologist (with video).

BACKGROUND AND AIMS: The prevalence and burden of ergonomic-related musculoskeletal injury are well established in the literature, but data are scarce on techniques that can be used to avoid injury. This pilot study aimed to develop a new method of endoscopist wellness assessment. The technique presented here is an intervention by a physical therapist assessing ergonomic position and posturing during endoscopy to create an individualized wellness plan. METHODS: Volunteer endoscopists were identified in a single ambulatory surgical center. Demographics, previous injury, current pain, and posture were evaluated. A comprehensive assessment was developed by the physical therapist while observing endoscopists performing at least 2 colonoscopies and while working at their computer workspace. The detailed personalized wellness program included recommendations for individualized exercises, static and dynamic posture re-education during and between procedures, optimization of procedure suite setup, pain education, and an opportunity for follow-up 1-on-1 sessions with the physical therapist. Endoscopists were later interviewed regarding their perception of and compliance with the wellness plan. Specific outcomes evaluated included changes in musculoskeletal pain, acceptance, and incorporation of wellness recommendations and procedure suite alterations into clinical practice. RESULTS: As we developed this new method of endoscopic wellness assessment, 8 endoscopists representing a wide range of ages and clinical experience were assessed. Twenty-two pain sites were identified among 5 subjects, with back and neck pain the most common pain sites. A variety of ergonomic inefficiencies and suboptimal movement patterns was observed, resulting in highly variant wellness plans. By the end of the study, 63% of pain sites were reduced in intensity or resolved, whereas 32% of pain sites were unchanged and 4% increased in intensity. Seven of 8 participants found the pictures depicting their posture that supported their movement analysis helpful, and 3 participants requested reassessment by the physical therapist. All participants reported static and dynamic postural education and procedure suite setup recommendations to be impactful to their ergonomic performance. CONCLUSIONS: Ergonomic assessment and instruction by a physical therapist was well received and resulted in improvement of musculoskeletal complaints among a cohort of endoscopists reporting baseline pain associated with performing endoscopy. In addition, this intervention provided ergonomic education that can be carried forward throughout their professional endoscopic career. We believe that ongoing individualized assessment and optimization of ergonomics is necessary because generalized wellness programs or even modifications to endoscopic equipment would not target all the unique ergonomic challenges faced by each physician. Ergonomic programs using the new method presented here could potentially contribute to career longevity, decrease burnout, reduce lost days of work, and, most importantly, reduce pain and fatigue among practitioners.

Lubiprostone protects esophageal mucosa from acid injury in porcine esophagus.

Esophageal injury from acid exposure related to gastroesophageal reflux disease is a common problem and a risk factor for development of Barrett's esophagus and esophageal adenocarcinoma. Our previous work highlights the benefits of using porcine esophagus to study human esophageal disease because of the similarities between porcine and human esophagus. In particular, esophageal submucosal glands (ESMGs) are present in human esophagus and proximal porcine esophagus but not in rodent esophagus. Although CFTR is expressed in the ducts of ESMGs, very little is known about CFTR and alternate anion channels, including ClC-2, in the setting of acid-related esophageal injury. After finding evidence of CFTR and ClC-2 in the basal layers of the squamous epithelium, and in the ducts of the ESMGs, we developed an ex vivo porcine model of esophageal acid injury. In this model, esophageal tissue was placed in Ussing chambers to determine the effect of pretreatment with the ClC-2 agonist lubiprostone on tissue damage related to acid exposure. Pretreatment with lubiprostone significantly reduced the level of acid injury and significantly augmented the recovery of the injured tissue (P < 0.05). Evaluation of the interepithelial tight junctions showed well-defined membrane localization of occludin in lubiprostone-treated injured tissues. Pretreatment of tissues with the Na+-K+-2Cl- cotransporter inhibitor bumetanide blocked lubiprostone-induced increases in short-circuit current and inhibited the reparative effect of lubiprostone. Furthermore, inhibition of ClC-2 with ZnCl2 blocked the effects of lubiprostone. We conclude that ClC-2 contributes to esophageal protection from acid exposure, potentially offering a new therapeutic target.NEW & NOTEWORTHY This research is the first to describe the presence of anion channels ClC-2 and CFTR localized to the basal epithelia of porcine esophageal mucosa and the esophageal submucosal glands. In the setting of ex vivo acid exposure, the ClC-2 agonist lubiprostone reduced acid-related injury and enhanced recovery of the epithelial barrier. This work may ultimately provide an alternate mechanism for treating gastroesophageal reflux disease.

Pathogenesis and Cells of Origin of Barrett's Esophagus.

In patients with Barrett's esophagus (BE), metaplastic columnar mucosa containing epithelial cells with gastric and intestinal features replaces esophageal squamous mucosa damaged by gastroesophageal reflux disease. This condition is estimated to affect 5.6% of adults in the United States, and is a major risk factor for esophageal adenocarcinoma. Despite the prevalence and importance of BE, its pathogenesis is incompletely understood and there are disagreements over the cells of origin. We review mechanisms of BE pathogenesis, including transdifferentiation and transcommitment, and discuss potential cells of origin, including basal cells of the squamous epithelium, cells of esophageal submucosal glands and their ducts, cells of the proximal stomach, and specialized populations of cells at the esophagogastric junction (residual embryonic cells and transitional basal cells). We discuss the concept of metaplasia as a wound-healing response, and how cardiac mucosa might be the precursor of the intestinal metaplasia of BE. Finally, we discuss shortcomings in current diagnostic criteria for BE that have important clinical implications.

Plasmonic nanobiosensors for detection of microRNA cancer biomarkers in clinical samples.

MicroRNAs (miRNAs) play an important role in the regulation of biological processes and have demonstrated great potential as biomarkers for the early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE), the premalignant metaplasia associated with EAC. Herein, we demonstrate the direct detection of the esophageal cancer biomarker, miR-21, in RNA extracted from 17 endoscopic tissue biopsies using the nanophotonics technology our group has developed, termed the inverse molecular sentinel (iMS) nanobiosensor, with surface-enhanced Raman scattering (SERS) detection. The potential of this label-free, homogeneous biosensor for cancer diagnosis without the need for target amplification was demonstrated by discriminating esophageal cancer and Barrett's esophagus from normal tissue with notable diagnostic accuracy. This work establishes the potential of the iMS nanobiosensor for cancer diagnostics via miRNA detection in clinical samples without the need for target amplification, validating the potential of this assay as part of a new diagnostic strategy. Combining miRNA diagnostics with the nanophotonics technology will result in a paradigm shift in achieving a general molecular analysis tool that has widespread applicability for cancer research as well as detection of cancer. We anticipate further development of this technique for future use in point-of-care testing as an alternative to histopathological diagnosis as our method provides a quick result following RNA isolation, allowing for timely treatment.

Inconsistencies in Colonic Tattooing Practice: Differences in Reported and Actual Practices at a Tertiary Medical Center.

OBJECTIVES: Accurate localization of a colonic lesion is crucial to successful resection. Although colonic tattooing is a widely accepted technique to mark lesions for future identification surgery or repeat colonoscopy, no consensus guidelines exist. The objective of this study was to determine whether the current tattooing practice at a tertiary medical center differs from recommendations in the literature and self-reported provider practice. METHODS: The study consisted of an observational retrospective chart review of patients who received colonic tattoos, as well as a provider survey of reported tattooing practices at a tertiary academic medical center. A total of 747 patients older than 18 years of age who underwent colonoscopy with tattoo were included. Forty-four gastroenterologists performing endoscopy were surveyed on tattooing techniques. RESULTS: In the majority of cases, neither the number of tattoos, location of the tattoo nor the distance from the lesion was specified within the report. Following the index procedure, a tattoo was detected in 75% of surgical resections and 73% of endoscopies. At the time of surgery, however, the tattoo and/or the lesion was detected approximately 94% of the time. Twenty-five endoscopists (56.8%) completed the survey. Differences were seen the between the chart review and reported practice. Most providers report placing ≥2 marks (87.2%); however, chart review revealed that only 56.2 % were tattooed with ≥2 marks. CONCLUSIONS: Variation exists between the reported tattooing practice and actual practice. Despite this, most tattoos are identified at the time of surgery or repeat endoscopy. Further research is needed to determine whether a standardized approach to tattooing and reporting could improve localization at repeat endoscopy.

Helicobacter pylori-associated peptic ulcer disease: A retrospective analysis of post-treatment testing practices.

BACKGROUND & AIMS: Guidelines recommend that patients with Helicobacter pylori (H. pylori)-associated peptic ulcer disease (PUD) receive H. pylori eradication therapy followed by post-treatment testing to prove eradication; however, post-treatment testing rates are suboptimal and barriers to testing are poorly understood. Our aim was to identify factors that predicted receipt of post-treatment testing. METHODS: We performed a retrospective cohort study of 152 patients with H. pylori-associated PUD diagnosed between 2007 and 2015 at a large tertiary medical center in the United States, who received standard eradication therapy and ambulatory follow-up within one year. The primary outcome of interest was receipt of post-treatment testing. Logistic regression models compared post-treatment testing rates in those diagnosed while outpatient vs inpatient, patients with vs without repeat endoscopy, and patients with vs without gastroenterology (GI) clinic follow-up. Propensity scores controlled for age, sex, race, ulcer location, and symptom persistence. RESULTS: Among 152 patients, 67 (44%) patients received post-treatment testing. There were significant differences in post-treatment testing rates in those diagnosed as outpatients vs inpatients (57% vs 33%; OR 3.87, P = 0.001) and in patients with vs without GI follow-up (62% vs 11%; OR 9.85, P < 0.0001). CONCLUSIONS: The rate of testing for eradication after treatment in patients with H. pylori- associated PUD was low. However, this was significantly improved in patients who have GI follow-up and whose diagnosis was made in the outpatient setting. Our study demonstrates a clear opportunity for quality improvement initiatives.

Ductular and proliferative response of esophageal submucosal glands in a porcine model of esophageal injury and repair.

Esophageal injury is a risk factor for diseases such as Barrett's esophagus (BE) and esophageal adenocarcinoma. To improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in the human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. To test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype, and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs, and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, and 72 h and 5 and 7 days following RFA and compared with control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs postinjury with a prominent population of SOX9-positive cells in ESMGs postinjury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair.NEW & NOTEWORTHY A novel porcine model of injury and repair using radiofrequency ablation has been developed, allowing for reproducible injury to the esophagus to study repair in an animal model with esophageal submucosal glands, a key anatomical feature and missing in rodent models but possibly harboring progenitor cells. There is a strong translational component to this porcine model given the anatomical and physiological similarities between pigs and humans.

Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.

OBJECTIVE: The ductular reaction (DR) involves mobilisation of reactive-appearing duct-like cells (RDC) along canals of Hering, and myofibroblastic (MF) differentiation of hepatic stellate cells (HSC) in the space of Disse. Perivascular cells in stem cell niches produce pleiotrophin (PTN) to inactivate the PTN receptor, protein tyrosine phosphatase receptor zeta-1 (PTPRZ1), thereby augmenting phosphoprotein-dependent signalling. We hypothesised that the DR is regulated by PTN/PTPRZ1 signalling. DESIGN: PTN-GFP, PTN-knockout (KO), PTPRZ1-KO, and wild type (WT) mice were examined before and after bile duct ligation (BDL) for PTN, PTPRZ1 and the DR. RDC and HSC from WT, PTN-KO, and PTPRZ1-KO mice were also treated with PTN to determine effects on downstream signaling phosphoproteins, gene expression, growth, and migration. Liver biopsies from patients with DRs were also interrogated. RESULTS: Although quiescent HSC and RDC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was demonstrated in healthy liver. BDL induced PTN in MF-HSC and increased PTPRZ1 in MF-HSC and RDC. In WT mice, BDL triggered a DR characterised by periportal accumulation of collagen, RDC and MF-HSC. All aspects of this DR were increased in PTN-KO mice and suppressed in PTPRZ1-KO mice. In vitro studies revealed PTN-dependent accumulation of phosphoproteins that control cell-cell adhesion and migration, with resultant inhibition of cell migration. PTPRZ1-positive cells were prominent in the DRs of patients with ductal plate defects and adult cholestatic diseases. CONCLUSIONS: PTN, and its receptor, PTPRZ1, regulate the DR to liver injury by controlling the migration of resident cells in adult liver progenitor niches.

Ductal metaplasia in oesophageal submucosal glands is associated with inflammation and oesophageal adenocarcinoma.

AIMS: Recent studies have suggested that oesophageal submucosal gland (ESMG) ducts harbour progenitor cells that may contribute to oesophageal metaplasia. Our objective was to determine whether histological differences exist between the ESMGs of individuals with and without oesophageal adenocarcinoma (EAC). METHODS AND RESULTS: We performed histological assessment of 343 unique ESMGs from 30 control patients, 24 patients with treatment-naïve high-grade columnar dysplasia (HGD) or EAC, and 23 non-EAC oesophagectomy cases. A gastrointestinal pathologist assessed haematoxylin and eosin-stained ESMG images by using a scoring system that assigns individual ESMG acini to five histological types (mucous, serous, oncocytic, dilated, or ductal metaplastic). In our model, ductal metaplastic acini were more common in patients with HGD/EAC (12.7%) than in controls (3.5%) (P = 0.006). We also identified greater proportions of acini with dilation (21.9%, P < 0.001) and, to a lesser extent, ductal metaplasia (4.3%, P = 0.001) in non-EAC oesophagectomy cases than in controls. Ductal metaplasia tended to occur in areas of mucosal ulceration or tumour. CONCLUSIONS: We found a clear association between ductal metaplastic ESMG acini and HGD/EAC. Non-EAC cases had dilated acini and some ductal dilation. Because ESMGs and ducts harbour putative progenitor cells, these associations could have significance for understanding the pathogenesis of EAC.

Promises and Limitations of Current Models for Understanding Barrett's Esophagus and Esophageal Adenocarcinoma.

BACKGROUND & AIMS: This review was developed to provide a thorough and effective update on models relevant to esophageal metaplasia, dysplasia, and carcinogenesis, focusing on the advantages and limitations of different models of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). METHODS: This expert review was written on the basis of a thorough review of the literature combined with expert interpretation of the state of the field. We emphasized advances over the years 2012-2023 and provided detailed information related to the characterization of established human esophageal cell lines. RESULTS: New insights have been gained into the pathogenesis of BE and EAC using patient-derived samples and single-cell approaches. Relevant animal models include genetic as well as surgical mouse models and emphasize the development of lesions at the squamocolumnar junction in the mouse stomach. Rat models are generated using surgical approaches that directly connect the small intestine and esophagus. Large animal models have the advantage of including features in human esophagus such as esophageal submucosal glands. Alternatively, cell culture approaches remain important in the field and allow for personalized approaches, and scientific rigor can be ensured by authentication of cell lines. CONCLUSIONS: Research in BE and EAC remains highly relevant given the morbidity and mortality associated with cancers of the tubular esophagus and gastroesophageal junction. Careful selection of models and inclusion of human samples whenever possible will ensure relevance to human health and disease.

Surface-Enhanced Raman Spectroscopy-Based Detection of Micro-RNA Biomarkers for Biomedical Diagnosis Using a Comparative Study of Interpretable Machine Learning Algorithms.

Surface-enhanced Raman spectroscopy (SERS) has wide diagnostic applications due to narrow spectral features that allow multiplex analysis. We have previously developed a multiplexed, SERS-based nanosensor for micro-RNA (miRNA) detection called the inverse molecular sentinel (iMS). Machine learning (ML) algorithms have been increasingly adopted for spectral analysis due to their ability to discover underlying patterns and relationships within large and complex data sets. However, the high dimensionality of SERS data poses a challenge for traditional ML techniques, which can be prone to overfitting and poor generalization. Non-negative matrix factorization (NMF) reduces the dimensionality of SERS data while preserving information content. In this paper, we compared the performance of ML methods including convolutional neural network (CNN), support vector regression, and extreme gradient boosting combined with and without NMF for spectral unmixing of four-way multiplexed SERS spectra from iMS assays used for miRNA detection. CNN achieved high accuracy in spectral unmixing. Incorporating NMF before CNN drastically decreased memory and training demands without sacrificing model performance on SERS spectral unmixing. Additionally, models were interpreted using gradient class activation maps and partial dependency plots to understand predictions. These models were used to analyze clinical SERS data from single-plexed iMS in RNA extracted from 17 endoscopic tissue biopsies. CNN and CNN-NMF, trained on multiplexed data, performed most accurately with RMSElabel = 0.101 and 9.68 × 10-2, respectively. We demonstrated that CNN-based ML shows great promise in spectral unmixing of multiplexed SERS spectra, and the effect of dimensionality reduction on performance and training speed.

Prevalence of the cagA Virulence Factor Varies by Race Among Helicobacter pylori -Infected Patients Undergoing Upper Endoscopy.

INTRODUCTION: We designed a race-conscious study to assess the presence of Helicobacter pylori v irulence factor cagA in a retrospective cohort of patients with active H. pylori infection. METHODS: We compared cagA status by race in gastric tissue samples from 473 patients diagnosed with active H. pylori infection from 2015 to 2019. RESULTS: H. pylori + Black patients were 2 times more likely to be cagA + than H. pylori + White patients (82% vs 36%, P < .0001). DISCUSSION: Presence of cagA is common among endoscopy patients with active H. pylori infection; appropriate testing and treatment of H. pylori can both reduce gastric cancer risk and address health disparities.

TNF Promoter Hypomethylation Is Associated With Mucosal Inflammation in IBD and Anti-TNF Response.

Background and Aims: Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the tumor necrosis factor (TNF) promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and nonresponders. Methods: We obtained mucosal biopsies from 200 participants (133 IBDs and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 nonresponders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated intestinal epithelial cells from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF nonresponders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed 2 missense variants in DNA methyltransferase 1, 1 of which had reduced function in vivo. Conclusion: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

TNF promoter hypomethylation is associated with mucosal inflammation in IBD and anti-TNF response.

Background and aims: Inflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders. Methods: We obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo. Conclusions: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.