Association of cagA-Positive Helicobacter pylori with MGMT Promoter Hypermethylation Across the Gastric Disease Spectrum and Gastric Cancer

Introduction

Gastric cancer continues to be a significant contributor to cancer morbidity and mortality, with Helicobacter pylori identified as a primary upstream factor in chronic gastritis and gastric carcinogenesis. In addition to inflammation, growing evidence suggests that epigenetic remodeling, especially DNA methylation of promoter regions, serves as a mechanistic link between infection and malignant transformation.^1,2 DNA methylation at CpG islands can turn off genes that stop tumors from growing and make it harder for the genome to stay stable. O-6-methylguanine-DNA methyltransferase (MGMT) is an important DNA repair enzyme that removes O-6 alkyl adducts.^3,4 When MGMT is hypermethylated at its promoter, it makes it harder for cells to repair DNA and increases the number of mutations. Virulence factors of H. pylori, particularly cagA, are biologically equipped to enhance host signaling and oxidative stress, promoting abnormal methylation in the gastric epithelium.^5,6 In high-burden contexts, elucidating the correlation between H. pylori (and CagA +/-) and MGMT promoter methylation across the clinical continuum from gastritis and peptic ulcer disease to lymphoma and adenocarcinoma is of immediate translational significance.^7,8

This study aims to elucidate the relationship between H. pylori infection, specifically CagA-positive strains, and MGMT promoter hypermethylation, as well as to assess the variability of methylation frequencies across standardized gastroduodenal diagnoses. The focus is on quantifying effect sizes for infection–methylation associations and contextualising methylation within the observed disease gradient, while also analyzing prevalent cofactors such as smoking, sex, residence, chronic illness, and treatment.⁹

The primary objective is to assess the correlation between H. pylori cagA positive and MGMT promoter hypermethylation within the gastroduodenal disease spectrum in an Iraqi cohort. The goals are: (i) to find out how common H. pylori, cagA carriage, and MGMT hypermethylation are in both control and patient groups; (ii) to use the right inferential statistics to find out how strong the link is between infection status and MGMT hypermethylation; (iii) to describe how MGMT methylation is spread across acute and chronic gastritis, peptic ulcer disease, gastric lymphoma, and gastric adenocarcinoma; (iv) to look into links between methylation status and key cofactors, pointing out variables that show strong links; and (v) to evaluate the potential of MGMT promoter hypermethylation as an epigenetic biomarker to help with risk stratification and early detection in endoscopy pathways.

Methods

Results

The study comprised 120 participants (40 controls and 80 patients). The sex distribution did not exhibit a significant difference between groups (females: 32.5% in controls vs 43.8% in patients; χ² = 1.406, p = 0.236; OR = 0.62, 95% CI 0.28–1.37). Patients were aged greater than controls (42.83 ± 13.49 vs 39.95 ± 12.59 years), but there are no significant differences between the two groups (χ² = 50.411, p = 0.125; OR = 2.33, 95% CI 1.04–5.22). There was a significant difference in residence by group: 50.0% of patients lived in rural areas compared to 30.0% of controls (χ² = 4.344, p = 0.037). The complementary comparison showed that patients were less likely to live in urban areas (χ² = 6.018, p = 0.014; OR = 0.38, 95% CI 0.17–0.83). Patients had a lower rate of smoking than controls (41.2% vs. 65.0%), but the difference was statistically significant (χ² = 13.125, p = 0.001). Patients were more likely to receive therapy (treated: 46.3% vs 27.5%; χ² = 3.906, p = 0.048; OR = 2.27, 95% CI 1.00–5.16). By design and clinical adjudication, all controls were free of gastroduodenal disease (100.0%), while all patients had a documented diagnosis (χ² = 120.0, p < 0.001) as reported in Table 1.

There was a clear difference in the number of infections and the number of virulence carriers between the groups. The prevalence of Helicobacter pylori was significantly higher in patients (72.5%) and there is no infection recorded in control (0%) with χ² = 56.129 and p < 0.00, indicating substantially elevated odds of infection in patients (OR = 210.60, 95% CI 12.42–3572.22). The cagA virulence gene was also found in more than half of the patients but not in any of the controls (56.3% vs 0%; χ² = 36.000, p < 0.001), which means That the effect size was very large (OR = 103.82, 95% CI 6.17–1747.38) as demonstrated in Figure 1 A+B.

Figure 1. Proportions of H. pylori (A) and cagA (B) positivity in controls (n = 40) and patients (n = 80).

There was a clear difference between the two groups: hypermethylation was present in 66.3% of patients and 0% of controls (χ² = 47.463, p < 0.001), which is a large effect size (OR = 157.58, 95% CI 9.33–2661.18). As reported in Table 2.

In a sample of 120 participants, sex, residence, chronic disease, and therapy exhibited no significant correlations with MGMT promoter methylation in bivariate analysis (all p > 0.10). The distribution of females versus males was similar between hypermethylated and unmethylated cases (χ² = 0.681, p = 0.409). The status of chronic disease did not vary with methylation (χ² = 0.510, p = 0.475). Residence indicated a non-significant trend towards increased hypermethylation in rural compared to urban environments (χ² = 2.239, p = 0.135). Previous treatment did not correlate with methylation status (χ² = 0.456, p = 0.499). Conversely, smoking exhibited a significant correlation with methylation (overall χ² with 3 categories = 15.218, p < 0.001), and the aggregated comparison revealed increased odds of hypermethylation among smokers compared to non-smokers (OR = 4.55, 95% CI 1.96–10.55) as showed in Table 3.

There was a significant association between H. pylori infection and MGMT promoter hypermethylation (Table 4). In H. pylori positive cases, 74.1% exhibited hypermethylation, whereas only 16.1% of H. pylori–negative cases showed this characteristic (χ² = 40.892, p < 0.001), indicating significantly elevated odds of hypermethylation associated with infection (OR = 14.91, 95% CI 6.09–36.60). The association was more significant for cagA positivity, with hypermethylation seen in 91.1% of cagA-positive people compared to 16.0% of cagA-negative people (χ² = 64.345, p < 0.001; OR = 53.81, 95% CI 16.24–178.31) as reported in Table 4.

Discussion

Helicobacter pylori infection significantly promotes the progression of chronic gastritis to gastric carcinoma by influencing inflammatory and oxidative pathways, as well as inducing epigenetic modification of host gastric epithelial cells. The present study has a cross-sectional, observational design and therefore cannot establish a definitive causal or mechanistic relationship between H. pylori, particularly cagA-positive strains, and MGMT promoter hypermethylation. The findings demonstrate H. pylori induced oxidative stress, DNMT upregulation, and promoter hypermethylation. Integrating insights that H. pylori act as an upstream driver of MGMT epigenetic silencing, but longitudinal and interventional studies are required to confirm this causal pathway. When tumor suppressors and DNA repair genes, like MGMT, get too much methylation, the DNA repair machinery stops working. This is the most important change.¹¹ This makes mutations build up and the cells become cancerous. recurrent inflammation, oxidative stress, and host signaling pathways are the main reasons why H. pylori cause MGMT to be epigenetically silenced. Studies show that when H. pylori invade the gastric mucosa, it triggers a strong immune response that damages DNA and turns on DNA damage response pathways.¹² This leads to an overexpression of DNA methyltransferases (DNMTs). These enzymes are very important because they move methyl groups to cytosine residues. This leads to unusual DNA methylation in the promoter areas of genes that control cell cycle arrest, apoptosis, and DNA repair. MGMT, a crucial DNA repair gene, is particularly susceptible to methylation-mediated silencing during H. pylori infection, promoting neoplastic progression.¹³ The detection of MGMT promoter hypermethylation in inflamed but histologically benign mucosa underscores its possible function as an early molecular event in gastric carcinogenesis. Longitudinal data suggest that the elimination of H. pylori infection can partially reverse or impede these methylation changes, indicating that bacterial eradication not only reduces inflammation but may also restore normal epigenetic regulation in the gastric mucosa.¹⁴ A number of studies suggest that MGMT promoter hypermethylation may serve as a non-invasive biomarker for risk assessment and early detection, potentially influencing therapeutic sensitivity and prognosis in cancers associated with MGMT silencing.¹⁵ Nonetheless, the strength of this relationship may vary among populations due to genetic, dietary, and environmental influences on methyl group metabolism and inflammatory response, supporting the conclusions of this study.^16,17 A study confirmed the results of this research, demonstrating that H. pylori colonisation correlates with elevated DNMT activity and a hypermethylation of islands of CpG in the gastric mucosa, with MGMT identified as one of the most frequently silenced DNA repair genes.¹⁸ A study confirmed these findings by showing that MGMT methylation can be detected not only in cancerous tissues but also in inflamed, non-neoplastic mucosa, corresponding with an epigenetic “field defect” that occurs prior to histological transformation. Another study confirmed the finding of this study that cagA positivity is linked to the strongest association. This study found that cagA-positive strains cause more significant promoter hypermethylation and greater suppression of MGMT expression than cagA-negative strains.¹⁹ A subsequent study validated the present findings by demonstrating that H. pylori-induced oxidative and inflammatory signaling elevates DNMT1 expression and encourages MGMT silencing, thereby hindering the repair of O6-alkylguanine adducts and promoting the accumulation of mutations. The gradient hypermethylation identified in gastritis and peptic ulcer disease, progressing to malignant outcomes, is supported by prior clinical studies.²⁰ A study validated the results of this research, illustrating progressive elevations in MGMT methylation from chronic gastritis to intestinal metaplasia and carcinoma.^21,22 This suggests that methylation acts as an early and enduring indicator of H. pylori-associated mucosal remodeling. A study confirmed a similar trend by showing that MGMT methylation often comes before morphological dysplasia, which supports its use as a risk-stratification marker during endoscopic surveillance.²³ Interventional data supports our etiological interpretation: a study confirmed the findings of this research and recorded a partial reversal or reduction of aberrant methylation following H. pylori eradication, suggesting that a segment of the MGMT methylation is infection-dependent and subject to modification. A study validated the results of this research demonstrating a heightened probability of MGMT hypermethylation in smokers, consistent with cumulative oxidative stress and methylation pressure.²⁴ A study also confirmed that dietary methyl donors, nitrosamine exposure, and polymorphisms in methylation or repair genes influence the variations in effect sizes across studies.²⁵ In addition to MGMT, a study confirmed the broader context and documented the coordinated methylation of additional tumor-suppressor and mismatch-repair loci in H. pylori–infected mucosa, situating MGMT within a more extensive, infection-induced epigenetic framework that undermines genome preservation and epithelial homeostasis.²⁶ The comparative evidence collectively supports a unified model in which H. pylori, especially the most cagA positive strains, act as an upstream catalyst for MGMT promoter hypermethylation during the initial phases of the gastritis carcinoma sequence. Studies validating these results indicate a persistent increase in MGMT methylation within infected, inflamed mucosa, its presence in precancerous phases, and a degree of reversibility following eradication.^26,27 In contrast, research that challenges these findings primarily focusses on late-stage, tumor-exclusive cohorts, where subsequent epigenetic remodeling may obscure the initial microbial signal. In this context, MGMT hypermethylation is recognized as an indicator of infection-related epigenetic damage and a potential biomarker for risk stratification in endoscopic procedures.²⁷ Standardized, quantitative methylation assays performed longitudinally across well-defined stages accounting for cagA status, sampling site, eradication history, and lifestyle cofactors are imperative to address existing discrepancies and to determine the clinical relevance of MGMT methylation in H. pylori-associated gastric carcinogenesis.²⁸ The extremely large ORs and wide CI for some comparisons (e.g., infection prevalence in cases vs controls) result from zero events in the control group. These estimates indicating the presence of a very strong association rather than providing an exact quantitative measure of risk.

In conclusion, the interaction of inflammatory, oxidative, and virulence-mediated mechanisms clarifies the strong link between H. pylori infection, especially with cagA-positive strains, and MGMT promoter hypermethylation in the gastric mucosa. This epigenetic alteration serves as a crucial connection between chronic infection and the molecular initiation of gastric carcinogenesis by disrupting an essential DNA repair mechanism. MGMT hypermethylation not only indicates the degree of damage inflicted by infection but also functions as a prognostic marker for malignant potential. Its frequent occurrence across diverse populations and disease stages underscores its generality as an epigenetic signature linked to infection. Comprehending and targeting this methylation pathway offers promising opportunities for early diagnosis, chemoprevention, and customised management of H. pylori-related gastric cancer.

Ethical considerations

This study was conducted in accordance with the ethical standards of the Iraqi Ministry of Health and the regulations of the Al-Anbar Health Directorate. Ethical approval was obtained from the Research Committee of the Al-Anbar Health Directorate (Approval No. 35813, dated 28 October 2025). In addition, ethical approval was granted by the Research Ethics Committee of the University of Fallujah, College of Applied Sciences (Approval No. AS-EC/0011, dated 21 December 2025). All procedures involving human participants were performed in accordance with the Declaration of Helsinki. Gastric biopsy samples were collected only after obtaining written informed consent from all participants. All personal identifiers were removed prior to data analysis to ensure participant confidentiality.