SPERMINE OXIDASE MEDIATES <i>HELICOBACTER PYLORI</i>-INDUCED GASTRIC CARCINOGENESIS

Background: Tools predicting incident esophageal adenocarcinoma (EAC) and gastric cardia adenocarcinoma (GCA) that can be automated in electronic health records (EHRs) to guide screening decisions are needed. However, EHRs often have missing data for smoking, body mass index (BMI), and gastroesophageal reflux disease (GERD) which are important factors used by currently validated tools (Trøndelag Health [HUNT] and Kunzmann). We aimed to accurately predict EAC/GCA even with missing data using machine learning.
Methods: We performed retrospective analyses in the Veterans Health Administration (VHA) Corporate Data Warehouse among Veterans with ≥1 encounter between 2005 and 2018. Cases diagnosed with EAC/GCA were identified in the VHA Central Cancer Registry. The index date was the date of diagnosis for cases and randomly selected for controls. We collected prescriptions, laboratory results, and International Classification of Diseases diagnoses 1 to 5 years prior to index. We randomly divided the cohort into training (50%), preliminary validation (25%), and testing (25%). In the preliminary validation set, simple random sampling imputation and extreme gradient boosting machine learning were most accurate. In the test set, we compared the final model, the Kettles Esophageal and Cardia Adenocarcinoma predictioN (K-ECAN) Tool, to HUNT, Kunzmann, and published guidelines. To simulate a non-VHA population, we randomly under-sampled males. We ranked the proportion of the total gain in the loss function and the mean Shapley Additive Explanations for each variable.
Results: We identified 8,430 cases of EAC, 2,965 of GCA, and 10,256,887 controls. The mean age was 59.6 years, 92% were male, 80% white, and mean BMI 29.3 kg/m². In the test set, K-ECAN was well calibrated (Figure 1) and had better discrimination (area under the receiver operating characteristics curve [AUC] = 0.77) than HUNT (AUC = 0.68), Kunzmann (AUC = 0.64), or guidelines (Figure 2). Using only data from 4-5 years prior to index slightly diminished its accuracy (AUC = 0.75). Under-sampling men to simulate a non-VHA population, the AUCs of HUNT and Kunzmann improved, but K-ECAN was still most accurate (AUC = 0.85, Figure 2). The most important variables influencing K-ECAN included 4 known risk factors (age, race, sex, BMI) and 9 novel (COPD, greater Hct, lower HDL, greater LDL, lower serum CO₂, lower Na, lower BUN, lower ALT, and greater WBC). While GERD was strongly associated with EAC, it only contributed a small proportion of gain in information.
Conclusions: We developed and internally validated a novel prediction tool for incident EAC/GCA using EHR data. K-ECAN identifies individuals who are at increased risk for EAC/GCA ≥ 3 years in advance and is more accurate than published guidelines. Further work is needed to validate K-ECAN outside VHA and to assess how best to implement it within EHRs.

Introduction: Eosinophilic esophagitis (EoE) is a rare, chronic disease characterized by infiltration of eosinophils (eos) into the esophagus and symptoms related to esophageal dysfunction, such as dysphagia. Benralizumab, an eos-depleting anti-IL-5Rα monoclonal antibody, reduced eos in the esophagus in smaller studies and may be effective in EoE.
Methods: MESSINA (NCT04543409) was a phase 3, randomized (1:1), double-blind, placebo-controlled study evaluating benralizumab 30 mg subcutaneously every 4 weeks. Patients were 12-65 years with symptomatic (i.e., dysphagia) and histologically active EoE (≥15 eos/high-power field [hpf] on ≥2 esophageal levels); stable standard-of-care therapies were allowed. Dual primary endpoints were proportion of patients with histologic remission (≤6 eos/hpf) and change from baseline (CFB) in Dysphagia Symptom Questionnaire (DSQ) score at Week 24. Key secondary endpoints included percentage CFB in peak tissue eos and absolute CFB in Eosinophilic Esophagitis Histology Scoring System (EoE-HSS) grade and stage scores and Endoscopic Reference Score (EREFS). Symptoms were also assessed using the Eosinophilic Esophagitis Daily Dysphagia Diary (EoE-3D) and the Patient Global Impression of Severity (PGI-S). Selected patients underwent a sub-study with additional endoscopies and biopsies at Weeks 4 & 12 to understand the time course and impact of tissue eos depletion.
Results: A total of 210 patients (103 benralizumab; 107 placebo) were randomized and received treatment. Baseline characteristics were balanced between groups. At Week 24, a significantly higher proportion of patients on benralizumab vs placebo achieved histologic remission (87.4% vs 6.5%; p<0.0001); however, there was no difference in CFB in DSQ score between groups (–12.1 vs –15.1; p=0.1770) (Figure 1). Nominal statistical significance was observed for benralizumab vs placebo across the histology-based secondary endpoints (percentage CFB in peak tissue eos and EoE-HSS grade and stage scores) but not in CFB in EREFS at Week 24 (Figure 2). There were no differences between treatment groups for symptoms as measured by the EoE-3D and PGI-S. The rate of adverse events was 64.1% for patients on benralizumab and 61.7% for placebo. Benralizumab was well tolerated with no new safety signals. Additionally, of the 25 sub-study patients (11 benralizumab; 14 placebo), a higher proportion of patients on benralizumab vs placebo achieved histologic remission, and they had a higher percentage CFB in peak tissue eos at Weeks 4 & 12 than placebo, which was maintained to Week 24 (Figure 2).
Conclusions: Benralizumab reduced esophageal eos at 4 weeks and maintained this reduction to 24 weeks without a significant improvement in symptoms for patients with EoE vs placebo, suggesting that eos are not the primary driver of disease activity and symptomology.

Background: Multiple rapid swallows (MRS) has been proposed as a provocative test performed during high resolution manometry (HRM) to assess esophageal peristaltic reserve. Prior studies have shown that the vigor of peristalsis during MRS is inversely correlated with acid exposure time in nonerosive reflux disease, however the association between peristaltic reserve and extraesophageal reflux has not been elucidated.

Aim: To assess the role of esophageal contractile reserve, or MRS response, in the evaluation of patients with laryngopharyngeal reflux (LPR) symptoms.

Methods: Adults who underwent HRM and multichannel-intraluminal impedance-pH testing (MII-pH) off proton pump inhibitors for evaluation of LPR symptoms at a tertiary care center from 6/2019-10/2021 were included. The magnitude of MRS response was calculated by the ratio of MRS DCI post-contractions divided by the mean DCI from a series of single water swallows in supine position (MRS:SS DCI ratio). An intact MRS response was defined as MRS:SS DCI ratio >1. The proportion of reflux episodes reaching proximal extent was defined as the number of proximal reflux episodes divided by total reflux episodes. Univariate analyses were performed using Spearman’s correlation. Multivariate analyses were performed using general linear regression.

Results: 70 patients (68.7% female, mean age 56.9±14.0 years) were enrolled. Intact MRS response significantly correlated with fewer proximal reflux episodes (17.0±13.4 vs 23.0±19.0, p=0.0347) and greater complete bolus transit on HRM with impedance (63.2% vs 41.2%, p=0.0028) compared to absent reserve. The mean MRS DCI was inversely associated with proximal reflux episodes (R=-0.291, p=0.025) and total reflux episodes (R=-0.281, p=0.031). The magnitude of MRS response also inversely correlated with proximal reflux episodes (R=-0.25, p=0.045) and proportion of reflux episodes reaching proximal extent (R=-0.291, p=0.025). On multivariate analyses adjusting for age, sex, body mass index, smoking, and percent of ineffective swallows, an intact MRS response remained independently associated with fewer proximal and total reflux episodes and greater complete bolus clearance (Figure 1A). The mean MRS DCI also independently correlated with fewer proximal and total reflux events, as well as lower proportion of reflux episodes reaching proximal extent (Figure 1B).

Conclusion:
An intact MRS response is independently associated with decreased proximal reflux and greater complete bolus transit. The magnitude of MRS response also correlated with decreased proximal reflux and lower proportion of reflux events reaching the proximal esophagus. Esophageal contractile reserve may contribute to the protective mechanism against proximal migration of refluxate by improved clearance. Provocative measures such as MRS may have relevance in the evaluation of patients with LPR symptoms.

Introduction: We have implicated polyamines and their metabolism during Helicobacter pylori infection in gastric cancer. Notably, the polyamine spermine is back-converted to spermidine (Spd) by spermine oxidase (SMOX) and generates H₂O₂ and the reactive aldehyde acrolein. We have shown that H. pylori infection increases SMOX expression in human and mouse tissues and that infected C57BL/6 Smox^–/– mice exhibit decreases in gastric Spd, gastritis, and β-catenin activation. Our goal was to determine if increased SMOX expression during H. pylori infection leads to gastric cancer development using a molecular approach.
Methods: C57BL/6 Smox^–/– mice were backcrossed to the FVB/N background and then crossed to transgenic FVB/N insulin-gastrin (INS-GAS) mice, which overexpress the human gastrin gene and develop dysplasia and intramucosal carcinoma (IMC) with H. pylori infection. WT and Smox^–/– INS-GAS mice were infected with H. pylori PMSS1 for 8 weeks. Mice were also given 14 mM Spd in their drinking water beginning 1 week after infection. Histology was analyzed by H&E staining. Immunohistochemistry for Ki67 and quantification (10 high power fields/mouse) were used to assess proliferation. Expression of RNA was quantified by real-time PCR. Concentrations of gastric polyamines (nmol/mg protein) and acrolein (pmol/mg protein) were assessed by LC/MS.
Results: We confirmed that Smox was not expressed in the stomachs of INS-GAS mice with Smox deletion (P=0.0059 vs. WT). In addition, spermidine levels in the gastric tissues of Smox^–/– INS-GAS mice were significantly reduced (11.6 ± 0.7; n=5) compared to WT mice (17.2 ± 0.7; P=0.0079). We found a significant reduction in the extent of dysplasia and cancer after 8 weeks in the infected Smox^–/– INS-GAS mice (18.6 ± 3.8%; n=29) compared to the infected WT mice (39.5 ± 6.2%; n=21; P=0.0083). Only 2 out of 29 infected Smox^–/– INS-GAS mice exhibited IMC (6.9%), compared to 11 out of 21 in the infected WT INS-GAS mice (52.4%; P=0.0012). In parallel, acrolein synthesis in infected INS-GAS mice was significantly reduced with Smox deletion (919.6 ± 87.2, n=3 vs. WT 1238 ± 56.3, n=3; P=0.0479). Spd supplementation did not reverse the phenotype seen in the Smox^–/– mice. Since Ki67 is strongly associated with tumor cell growth, we assessed its expression and found that infected Smox^–/– INS-GAS mice displaying LGD exhibited less positive Ki67 epithelial cell nuclear staining (271.9 ± 18.6; n=5) compared to infected WT animals with IMC (380.0 ± 41.2; n=3; P=0.0113).
Conclusion: We observed that the deletion of Smox prevents acrolein synthesis and IMC development in H. pylori-infected INS-GAS mice. We envision novel therapies to alter SMOX-driven dysregulation of polyamine metabolism and thus reduce the burden of chronic gastritis progression to gastric cancer in H. pylori-infected patients.