ABSTRACT
Background
Helicobacter pylori infection is a significant contributing factor of gastric cancer. Metachronous neoplasms also pose a risk. The mechanism underlying the impact of H. pylori eradication on preventing metachronous gastric cancer is unclear. This study aimed to investigate immunity changes in gastric mucosa after H. pylori eradication and to identify mechanisms preventing metachronous recurrence.
Materials and Methods
Patients diagnosed with gastric neoplasm and H. pylori infection, who underwent endoscopic resection, were included. Thirty‐six cases of metachronous neoplasms occurring after eradication (metachronous group) were compared to 36 controls matched for age, sex, atrophy, and metaplasia (control group). Histological features and immunohistochemical staining for T‐cell (CD3, CD4, and CD8) and immune exhaustion (forkhead/winged helix transcription factor and programmed cell death‐ligand 1) markers in the non‐tumor‐bearing mucosa were evaluated.
Results
In histologic features, glandular atrophy and intestinal metaplasia in the gastric mucosa significantly improved following H. pylori eradication in the control group (p < 0.001, 0.008), whereas they did not improve in the metachronous group (p = 0.449, 0.609). CD8 and CD8/CD3 ratios increased in the control group (p < 0.001, 0.04), but did not show differences in the metachronous group (p = 0.057, 0.245). The CD4/CD3 ratio and programmed cell death‐ligand 1/CD4 expression significantly decreased after H. pylori eradication in the control group (p = 0.003, 0.042), but not in the metachronous group (p = 0.54, 0.55).
Conclusions
This observational study suggests that H. pylori eradication may prevent the recurrence of gastric neoplasia by improving histological inflammation and overcoming immune exhaustion.
Keywords: endoscopic submucosal dissection, gastric cancer, Helicobacter infections, immune system
Abbreviations
- Foxp3
forkhead/winged helix transcription factor
- H. pylori
Helicobacter pylori
- PD‐L1
programmed cell death‐ligand 1
1. Introduction
Gastric cancer is the third leading cause of cancer‐related death globally [1]. It is a multifactorial disease, with Helicobacter pylori ( H. pylori ) infection identified as the strongest risk factor [2]. H. pylori infects approximately half of the world's population and is considered an initial trigger in the Correa cascade, a model of gastric carcinogenesis that progresses from superficial gastritis through atrophic gastritis, intestinal metaplasia, and dysplasia, leading to adenocarcinoma [2, 3, 4]. Chronic H. pylori infection can also cause genetic instability in the gastric mucosa through a persistent active immune response that lasts throughout the lifespan of the host if the infection is not eradicated [5, 6, 7].
Advances in endoscopic techniques have facilitated the treatment of many cases of early gastric cancer with endoscopic resection; however, in contrast to surgery, it leaves most of the stomach intact, raising concerns regarding metachronous recurrence [8]. H. pylori eradication after endoscopic resection for early gastric cancer is recommended to reduce the risk of incidence of gastric neoplasms [9, 10, 11]. However, the mechanism by which H. pylori eradication prevents metachronous recurrence remains unclear.
Prolonged antigen exposure because of chronic infection leads to the progressive loss of T‐cell proliferation, cytokine production, and cytotoxic activity [12]. The immune exhaustion of T cells, marked by the upregulation of inhibitory receptors such as programmed cell death 1 (PD‐1) and the increased presence of regulatory T cells (Tregs), plays a crucial role in the immune evasion mechanisms of various cancers [13, 14, 15]. This exhausted state of T cells impairs the body's ability to control infections and facilitates the development and progression of tumors by allowing cancer cells to evade immune surveillance. Thus, understanding and reversing T‐cell exhaustion is critical for improving immune responses in chronic infections and cancer therapy.
PD‐1 is a protein expressed on the surface of T cells, B cells, and natural killer cells. PD‐1 binds to programmed cell death‐ligand 1 (PD‐L1), inducing T‐cell exhaustion and promoting the differentiation of regulatory T cells, which affects the prognosis of gastric cancer [16, 17, 18]. Additionally, among the immunological factors involved in gastric cancer development, Tregs, characterized by the expression of the forkhead/winged helix transcription factor (Foxp3), contribute to carcinogenesis by inhibiting CD8+ cytotoxic T‐cells [19, 20].
Therefore, we hypothesized that H. pylori eradication after endoscopic resection of early gastric cancer may help prevent metachronous recurrence by recovering immune exhaustion in the gastric mucosa, which could eliminate potential tumor antigens. By comparing the expression levels of exhausted T‐cells and Tregs in the surrounding gastric mucosa before and after H. pylori eradication, we aimed to investigate the mechanism by which H. pylori eradication reduces the risk of metachronous recurrence.
2. Methods
2.1. Patients
This 1:1 matched case–control study included patients who underwent endoscopic resection because of a gastric neoplasm, including dysplasia and adenocarcinoma, and with identified H. pylori infection at a single center, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea, between January 2010 and December 2019. The inclusion criteria were patients with confirmed H. pylori infection at the time of the first endoscopic resection who subsequently received eradication treatment. Exclusion criteria included patients with no confirmed H. pylori infection ever, prior eradication therapy before the first endoscopic resection, failed eradication, or successful eradication that is not confirmed via rapid urease test or urea breath test, inadequate tissue sample preservation, or follow‐up periods shorter than 3 months after eradication.
During this period, a total of 2590 patients underwent gastric endoscopic resection for gastric neoplasms, including low‐ and high‐grade epithelial dysplasia and adenocarcinoma. After reviewing records that followed up these patients through December 2023, we found that 236 patients underwent endoscopic resection for recurrence of gastric neoplasm. From these patients, we selected those who had confirmed H. pylori infection during the first endoscopic resection and received eradication treatment. After H. pylori eradication and confirmation with a negative result on rapid urease test or urea breath test, 41 patients showed development of metachronous gastric neoplasm (dysplasia or adenocarcinoma) after the first endoscopic resection for a previous gastric neoplasm and were treated by a second endoscopic resection for a new lesion. We categorized them as the metachronous group. Forty‐one patients matched by age, sex, and the grade of atrophy and intestinal metaplasia were enrolled and categorized as the control group. The control group comprised patients who underwent endoscopic resection for gastric neoplasm but did not show any metachronous gastric neoplasm during the follow‐up period.
We defined metachronous lesions as those appearing at a different site at least 6 months after the previous endoscopic resection. The interval between H. pylori eradication and the development of metachronous gastric neoplasms was at least 6 months, allowing for an accurate evaluation of the effect of H. pylori eradication on the incidence of metachronous lesions after the previous endoscopic resection. Figure 1 provides a brief flowchart of this study.
FIGURE 1.
Flowchart of the study. H. pylori , Helicobacter pylori .
This study was approved by the Yonsei University College of Medicine Institutional Review Board (IRB No. 3‐2023‐0325). The requirement for individual informed consent was waived because of the retrospective design of the study.
2.2. Endoscopic Procedure
Endoscopic resections were performed using standard techniques, including endoscopic submucosal dissection, by experienced gastroenterologists. The procedures were performed using high‐definition endoscopes equipped with narrow‐band imaging capabilities. The equipment used included the Olympus GIF‐H260, GIF‐2TQ260M, and GIF‐H290 series, Olympus corporation, Tokyo, Japan, which provide enhanced visualization of the mucosal and vascular patterns.
Endoscopic submucosal dissections were conducted using a dual knife with a 1.5‐mm cutting knife (KD‐650Q; Olympus, Tokyo, Japan). Injection solution, which consisted of a mixture of glycerol, indigo carmine, and epinephrine, was used to elevate the mucosa. Endoscopic submucosal dissection procedures aimed to achieve en bloc resection to ensure complete removal of lesions with a margin of healthy tissue.
Patients underwent conscious sedation with midazolam, propofol, and pethidine, and all procedures were monitored by an assistant doctor. The resected specimens were pinned on a cork board and fixed in 10% buffered formalin for histopathological examination.
2.3. Histological/Immunological Evaluation
Representative paraffin‐embedded sections from endoscopic submucosal dissection or biopsy tissues were selected for histological evaluation and immunohistochemical staining. The histological features of the gastric mucosa were assessed using 4‐μm‐thick hematoxylin and eosin‐stained slides and graded according to the updated Sydney system scores. One expert pathologist evaluated the degree (0: none, 1: mild, 2: moderate, and 3: severe) of chronic inflammation, neutrophil infiltration, atrophy, and intestinal metaplasia infiltration on each pathology slide [21, 22].
Immunohistochemical staining was conducted to detect T‐cell markers (CD3, CD4, and CD8) and immune exhaustion markers (Foxp3 and PD‐L1; Figure 2). The antibodies used are detailed in Table S1. All immunohistochemical analyses were performed on 4‐μm‐thick formalin‐fixed paraffin‐embedded tissue sections using an automated immunohistochemistry staining device (BenchMark XT; Ventana Medical Systems, Tucson, AZ, USA).
FIGURE 2.
Representative images of metachronous recurrent and control cases and immunohistochemical expression slides (hematoxylin and eosin, CD3, CD4, and CD8, 100× magnification). In the recurrent cases, minimal changes were observed in immune cell populations, aside from an increase in CD3 cells. In contrast, the control cases showed a significant increase in immune cells following eradication.
Expression levels were quantified using 3DHistech software (Budapest, Hungary)—NuclearQuant. It is a color‐ and pattern‐based image analysis solution designed to identify different tissue elements in stained samples. Rather than relying on time‐consuming manual annotation, the software allows for automated detection and annotation of regions of interest, following a brief training on relevant tissue samples. To avoid misquantification, only areas within the lamina propria, excluding epithelial glands, were annotated, ensuring that the annotated regions consisted solely of inflammatory cells. The annotated areas included regions of the lamina propria that are larger than a specified size: on average, 0.35 mm2 for endoscopic submucosal dissection samples and 0.2 mm2 for biopsy specimens. We used the proportion obtained by dividing the counted cells by the annotated area (Figure 3).
FIGURE 3.
Methods for quantification of immunologic markers. Immune cells are automatically counted by NuclearQuant in the annotated areas of the lamina propria. Red circles indicate the positive immunostained (brown color) CD4 cells, whereas blue circles indicate the negative cells.
2.4. Statistical Analysis
The baseline characteristics, except age, lesion size, and follow‐up interval, were categorical variables and are presented as mean and standard deviation. All variables of histologic analysis were categorical and those of immunologic expression level were continuous. Chi‐squared test and Fisher's exact test were employed to compare categorical variables of clinicopathological factors between groups based on the development of metachronous gastric neoplasms. For non‐categorical variables in intergroup comparisons of clinicopathological characteristics, the t‐test was used. To evaluate immunohistochemical expression levels, the Wilcoxon signed‐rank test and Mann–Whitney U test were applied. Sub‐group analysis was conducted by classifying the study participants into various sub‐groups based on criteria such as baseline characteristics (age and sex), whether the pathology of the primary lesion was dysplasia or adenocarcinoma, and whether the pathology of the metachronous recurrent lesion was dysplasia or adenocarcinoma. When comparing immunologic expression levels, we attempted both pre‐ and post‐eradication comparisons within the same patient as well as comparisons between different groups. For sub‐group analysis based on recurrent lesions, only the matched control groups in a 1:1 ratio were extracted and analyzed.
All statistical analyses were conducted using IBM SPSS Statistics for Windows, version 28.0 (IBM Corp., Armonk, NY, USA). A p‐value of less than 0.05 was considered statistically significant.
3. Results
3.1. Baseline Characteristics
Of the 40 patients in the metachronous group, four were excluded because of tissue‐sample‐preservation abnormalities that precluded analysis, thus including a total of 36 patients in the study. Matched patients in the control group were also excluded, resulting in a total of 72 patients included in the two groups.
Table 1 shows the comparison of various clinical characteristics between the two groups. Age and sex showed no significant differences between the groups because we matched them when enrolling patients. The average lesion size, lesion location, gross appearance, and dysplasia/adenocarcinoma rates were not significantly different (p = 0.883, 0.06, and 0.116, respectively). The recurrence interval in the metachronous group was 1055.3 days, and the pathology follow‐up interval in the control group was 579.4 days.
TABLE 1.
Baseline clinicopathological characteristics of patients in two groups.
| Variables | Metachronous group (n = 36) | Control group (n = 36) | p |
|---|---|---|---|
| Male (n, %) | 25 (69.4) | 25 (69.4) | — |
| Age (years, mean ± standard deviation) | 68.0 ± 7.8 | 64.6 ± 10.5 | 0.199 |
| Size (mm) | 18.9 ± 12.8 | 19.1 ± 12.9 | 0.883 |
| Location (n, %) | 0.06 | ||
| Upper 1/3 | 4 (11.1) | 1 (2.8) | |
| Mid 1/3 | 10 (27.8) | 6 (16.6) | |
| Lower 1/3 | 22 (61.1) | 29 (80.6) | |
| Gross appearance (n, %) | 0.116 | ||
| Elevated | 13 (36.1) | 6 (16.6) | |
| Flat | 13 (36.1) | 19 (52.8) | |
| Depressed | 10 (27.8) | 11 (30.6) | |
| Dysplasia/adenocarcinoma (n, %) | 7 (19.4)/29 (80.6) | 6 (16.6)/30 (83.4) | |
| Recurrence interval (days) | 1055.3 ± 809.7 | ||
| Pathology follow‐up interval (days) | 579.4 ± 314.5 | ||
3.2. Histologic Change After H. pylori Eradication
On comparison of the pre‐ and post‐eradication histological features between the metachronous and control groups, both groups showed significant improvement in chronic inflammation and neutrophil infiltration post‐eradication (p = 0.006 and 0.031 in the metachronous group, and p < 0.001 for both measures in the control group). In contrast, no significant change was observed in glandular atrophy and intestinal metaplasia scores post‐eradication in the metachronous group (p = 0.449 and 0.609, respectively). However, the control group showed significant improvement in these scores (p < 0.001 and p = 0.008, respectively; Table 2).
TABLE 2.
Changes in histologic features following Helicobacter pylori eradication.
| Variables | Metachronous (n = 36) | Control (n = 36) | ||||
|---|---|---|---|---|---|---|
| Pre‐eradication (n, %) | Post‐eradication (n, %) | p | Pre‐eradication (n, %) | Post‐eradication (n, %) | p | |
| Chronic inflammation | 0.006 | < 0.001 | ||||
| No | 0 (0) | 0 (0) | 1 (2.8) | 6 (16.7) | ||
| Mild | 8 (22.2) | 19 (52.8) | 5 (13.9) | 26 (72.2) | ||
| Moderate | 19 (52.8) | 14 (38.9) | 17 (47.2) | 4 (11.1) | ||
| Severe | 9 (25) | 3 (8.3) | 13 (36.1) | 0 (0) | ||
| Neutrophil infiltration | 0.031 | < 0.001 | ||||
| No | 10 (27.8) | 19 (52.8) | 10 (27.8) | 26 (72.2) | ||
| Mild | 20 (55.6) | 15 (41.6) | 16 (44.4) | 9 (25) | ||
| Moderate | 6 (16.6) | 2 (5.6) | 9 (25) | 0 (0) | ||
| Severe | 0 (0) | 0 (0) | 1 (2.8) | 1 (2.8) | ||
| Glandular atrophy | 0.449 | < 0.001 | ||||
| No | 3 (8.3) | 4 (11.1) | 3 (8.3) | 20 (55.6) | ||
| Mild | 19 (52.8) | 21 (58.3) | 23 (63.9) | 16 (44.4) | ||
| Moderate | 14 (38.9) | 10 (27.8) | 10 (27.8) | 0 (0) | ||
| Severe | 0 (0) | 1 (2.8) | 0 (0) | 0 (0) | ||
| Intestinal metaplasia | 0.609 | 0.008 | ||||
| No | 0 (0) | 0 (0) | 6 (16.6) | 11 (30.6) | ||
| Mild | 12 (33.4) | 16 (44.4) | 10 (27.8) | 16 (44.4) | ||
| Moderate | 16 (44.4) | 11 (30.6) | 15 (41.6) | 8 (22.2) | ||
| Severe | 8 (22.2) | 9 (25) | 5 (14.0) | 1 (2.8) | ||
Note: significance: p < 0.05.
3.3. Immunologic Change After H. pylori Eradication
We compared T‐cell‐ (CD3, CD4, and CD8) and immune exhaustion‐ (Foxp3 and PD‐L1) marker‐expression levels and their ratio before and after eradication in metachronous and control groups (Table 3). In both groups, CD3 showed a statistically significant increase after eradication (p = 0.017 and 0.022, respectively). However, in the metachronous group, other T‐cell‐ and immune exhaustion‐marker‐expression levels and their ratio showed no significant change between before and after eradication. On the other hand, in the control group, CD3 and CD8 expressions increased after eradication (p = 0.022 and < 0.001), and CD4/CD3, CD8/CD4, PD‐L1/CD4 ratios changed significantly after eradication (p = 0.003, 0.04, and 0.042, respectively).
TABLE 3.
Comparison of positive cell proportions of CD3, CD4, CD8, Foxp3, and PD‐L1 and their ratios before and after Helicobacter pylori eradication.
| Variables | Metachronous (n = 36) | Control (n = 36) | ||||
|---|---|---|---|---|---|---|
| Pre‐eradication | Post‐eradication | p | Pre‐eradication | Post‐eradication | p | |
| CD3 | 21.33 ± 9.78 | 26.71 ± 11.19 | 0.017 | 21.00 ± 12.07 | 27.59 ± 10.43 | 0.022 |
| CD4 | 16.82 ± 14.12 | 20.51 ± 10.90 | 0.109 | 17.65 ± 12.21 | 15.50 ± 8.81 | 0.194 |
| CD8 | 12.91 ± 7.06 | 14.94 ± 6.93 | 0.057 | 11.71 ± 5.52 | 19.44 ± 8.89 | < 0.001 |
| Foxp3 | 2.41 ± 2.42 | 2.71 ± 2.28 | 0.338 | 2.40 ± 2.60 | 2.06 ± 1.45 | 0.936 |
| PD‐L1 | 0.57 ± 1.21 | 0.31 ± 0.48 | 0.799 | 0.67 ± 1.36 | 0.28 ± 0.71 | 0.092 |
| CD4/CD3 | 0.79 ± 0.56 | 0.79 ± 0.35 | 0.54 | 0.79 ± 0.39 | 0.57 ± 0.26 | 0.003 |
| CD8/CD3 | 0.65 ± 0.31 | 0.59 ± 0.21 | 0.245 | 0.65 ± 0.35 | 0.76 ± 0.37 | 0.04 |
| PD‐L1/CD4 | 0.05 ± 0.14 | 0.02 ± 0.04 | 0.55 | 0.04 ± 0.07 | 0.02 ± 0.03 | 0.042 |
Abbreviations: Foxp3, forkhead/winged helix transcription factor; PD‐L1, programmed cell death‐ligand 1.
Note: significance: p < 0.05.
3.4. Metachronous Cancer‐Group Analysis
In the sub‐group analysis, we separated only cases in which the pathology of the recurrent lesion was adenocarcinoma and analyzed the expression levels between the two groups before and after eradication. After eradication, we found that the expression of CD8 was statistically higher in the control group, and the PD‐L1 and PD‐L1/CD4 ratio were significantly lower (Table 4; p = 0.033, 0.015, and 0.039, respectively).
TABLE 4.
Sub‐group analysis of immunologic markers in recurrent cases before and after Helicobacter pylori eradication.
| Variables | Pre‐eradication (n = 21, each) | Post‐eradication (n = 21, each) | ||||
|---|---|---|---|---|---|---|
| Recurrence | Control | p | Recurrence | Control | p | |
| CD3 | 18.47 ± 10.29 | 20.14 ± 12.40 | 0.394 | 24.20 ± 10.85 | 29.15 ± 10.94 | 0.217 |
| CD4 | 18.12 ± 14.30 | 17.71 ± 10.78 | 0.986 | 17.00 ± 9.68 | 17.24 ± 9.82 | 0.848 |
| CD8 | 11.08 ± 6.84 | 11.88 ± 6.26 | 0.639 | 13.72 ± 6.16 | 19.55 ± 9.09 | 0.033 |
| Foxp3 | 2.42 ± 2.79 | 2.36 ± 1.85 | 0.986 | 2.14 ± 2.22 | 2.15 ± 1.68 | 0.903 |
| PD‐L1 | 0.86 ± 1.52 | 0.76 ± 1.32 | 0.557 | 0.44 ± 0.58 | 0.26 ± 0.82 | 0.015 |
| CD4/CD3 | 0.98 ± 0.60 | 0.88 ± 0.30 | 0.59 | 0.74 ± 0.37 | 0.59 ± 0.28 | 0.149 |
| CD8/CD3 | 0.69 ± 0.38 | 0.69 ± 0.42 | 0.931 | 0.59 ± 0.20 | 0.72 ± 0.35 | 0.192 |
| PD‐L1/CD4 | 0.07 ± 0.18 | 0.06 ± 0.09 | 0.913 | 0.03 ± 0.04 | 0.01 ± 0.03 | 0.039 |
Abbreviations: Foxp3, forkhead/winged helix transcription factor; PD‐L1, programmed cell death‐ligand 1.
Note: significance: p < 0.05.
4. Discussion
H. pylori eradication in patients who have undergone endoscopic resection for gastric cancer and dysplasia reduces the risk of metachronous recurrence, but the mechanism remains unclear [23]. This study is the first to demonstrate that the immune system, exhausted by H. pylori infection and tumors, can recover after eradication, thereby preventing metachronous recurrence. Our study provides additional insights into the immunological mechanisms underlying the beneficial effects of eradication. In this 1:1 age, sex, grade of atrophy, and metaplasia matched case–control study, we observed significant immunohistological changes in the gastric mucosa following H. pylori eradication, particularly noting that metachronous recurrence of gastric neoplasm after endoscopic resection was associated with a failure to recover from immune exhaustion. The increase in CD8+ T‐cell expression in the control group, which did not exhibit recurrence during the follow‐up period after eradication, suggests that restoring T‐cell immunity from an exhausted state due to chronic infection may be linked to preventing metachronous recurrence.
CD8+ T‐cells are cytotoxic T lymphocytes that produce and express αβ‐T‐cell receptors with CD8 in the thymus [24]. They are pivotal in the immune system for eliminating tumor antigens. CD8+ T‐cells combat viruses, tumors, and other pathogens by recognizing and destroying cells presenting major histocompatibility complex class‐I molecules [25]. Chronic inflammation resulting from persistent infection, such as H. pylori infection, leads to a reduction in T‐cell proliferation and their ability to capture pathogens, ultimately causing CD8+ T‐cell exhaustion [14, 24]. This exhaustion is a known factor in the development and poor prognosis of gastrointestinal tract cancers, including gastric cancer, because of the decreased anti‐tumor immune function [14]. While the functional impairment of antigen‐specific T‐cells is a defining characteristic of many chronic infections, the underlying mechanisms of T‐cell dysfunction and repair after eradication are still unclear [26]. In our study, the patients who showed increased CD8 expression in the gastric tissue significantly after H. pylori eradication did not show metachronous recurrence of gastric neoplasm after previous endoscopic resection. This result suggests that restoring T‐cell immunity from an exhausted state because of chronic infection may be linked to preventing metachronous recurrence of gastric neoplasm after H. pylori eradication.
Several studies have focused on reversing T‐cell exhaustion during chronic infection and cancer. A major area of interest is the PD‐1/PD‐L1 pathway, which is selectively upregulated in exhausted T‐cells [12, 26]. Research has shown that blocking this inhibitory pathway can restore the functions of exhausted CD8+ T‐cells [27, 28, 29]. PD‐L1, a well‐known immune checkpoint protein, is expressed on the surface of cancer and stromal cells such as antigen‐presenting cells and activated T‐cells [30, 31]. PD‐L1 expression on memory CD4+ T‐cells activates regulatory T‐cells, which suppress the activity of naive T‐cells, reducing the immune response [13]. This mechanism promotes self‐tolerance and suppresses neighboring macrophages and effector T‐cells in cancer. A previous study has found that PD‐L1 expression in gastric cancer is stronger in the immune stroma than in the tumor, suggesting that PD‐L1 may affect the surrounding microenvironment and contribute to cancer development [32]. Based on this mechanism and previous findings, we analyzed the expression level of PD‐L1 and the PD‐L1/CD4 ratio in our study. While no statistically significant difference was observed, PD‐L1 expression decreased more in the control group after eradication, and the PD‐L1/CD4 ratio significantly decreased only in the control group after eradication. This suggests that the decrease in PD‐L1 expression on CD4+ T‐cells may contribute to the reactivation of exhausted CD8+ T‐cells.
In this study, we observed significant changes in the CD4/CD3 and CD8/CD3 ratios following H. pylori eradication, which provide critical insights into the immune dynamics within the gastric mucosa. We chose the ratio of CD4/CD3 because we considered that it effectively reflects the balance of helper T‐cells within the total T‐cell population, serving as an indicator of immune regulatory functions. A decrease in this ratio after eradication, observed in the control group, may signify a reduction in Tregs activity, potentially shifting the immune environment towards enhanced cytotoxicity and tumor suppression [33].
Conversely, the CD8/CD3 ratio represents the proportion of cytotoxic T‐cells, which is essential for directly targeting and eliminating cancerous cells. The significant increase in the CD8/CD3 ratio in the control group after eradication suggests a restoration of cytotoxic T‐cell function, highlighting the critical role of these cells in maintaining effective immune surveillance and preventing metachronous recurrence [33]. These findings underscore the importance of restoring immune balance and enhancing cytotoxic responses through H. pylori eradication, contributing to the overall reduction in the recurrence of gastric cancer.
Despite initial expectations that Foxp3 expression would differ significantly between the metachronous recurrence and control groups, our findings did not reveal any significant differences. Foxp3, a key marker for Tregs, plays a crucial role in maintaining immune tolerance and suppressing anti‐tumor immune responses [15]. Previous studies have highlighted the association between increased Foxp3+ Treg infiltration and poorer prognosis in various cancers, including gastric cancer [20, 34, 35]. Further, Foxp3‐positive cells may have an important role in severe H. pylori ‐associated gastritis [36]. However, in our study, the Foxp3 levels remained relatively unchanged between the two groups, both before and after H. pylori eradication. The lack of statistically significant results may not be because of the minimal role of Treg cells, but rather owing to the insufficient number of Foxp3‐positive cells, making sensitive analysis by immunohistochemistry challenging. Accurate assessment of Treg cells requires fluorescence activated cell sorter analysis, and further studies using this method may be necessary to elucidate the role of Tregs clearly.
The results of the sub‐group analysis, which focused on cases where the recurrent lesion was confirmed to be adenocarcinoma, supported the previous conclusions more strongly. We aimed to determine whether a direct comparison of immune marker expression before and after eradication between the control and recurrent adenocarcinoma sub‐groups would reveal any differences in cases that developed more severe metachronous lesions during the same follow‐up period. The analysis showed that immune markers did not differ between the recurrent and control groups before eradication. However, after eradication, CD8 expression was significantly higher in the control group, while PD‐L1 expression and the PD‐L1/CD4 ratio were significantly lower. We speculate that the activation of the immune system by reducing PD‐L1 expression and increasing CD8+ T‐cells has potential in preventing recurrence, indicating a potential pathway for enhancing treatment outcomes.
The two groups also showed significant differences in the recovery of glandular atrophy and intestinal metaplasia after eradication. Chronic inflammation and neutrophil infiltration were significantly reversed in both groups, but atrophy and intestinal metaplasia were reversed in the control group and not in the metachronous recurrence group. Several studies have explored whether H. pylori eradication can reverse atrophic gastritis and intestinal metaplasia, although the results have been controversial [37, 38, 39]. The findings of this study not only support previous research findings indicating that reversible changes in atrophic gastritis and intestinal metaplasia are associated with the suppression of gastric neoplasm recurrence after H. pylori eradication but also suggest that restoration of T‐cell immunity may be linked to these reversible changes. However, the causal relationship remains unclear.
The limitations of this study include its retrospective nature and relatively small sample size, which may affect the generalizability of the findings. Although we matched patients for key variables such as age, sex, and the grade of atrophy and intestinal metaplasia, other confounding factors might have influenced the outcomes. Moreover, the specific factors and mechanisms involved in the recovery of exhausted immunity after eradication remain unclear. Given the retrospective, observational design of our study, we could not definitively establish causality. Future prospective studies including tracking immune changes following H pylori infection and eradication are critical to validate the mechanistic pathways suggested by our clinical observations. Additionally, the role of PD‐L1 reduction in normal gastric immunity is still unclear. Future studies are needed to determine which specific cell populations express PD‐L1 within the gastric mucosa and to use functional assays evaluating whether PD‐L1 downregulation directly enhances CD8+ T‐cell activity in order to clarify these mechanisms.
Despite these limitations, our study has several important strengths that contribute to its value in the field of gastric neoplasms. To our knowledge, this is the first study to identify an association between T‐cell exhaustion and the occurrence of metachronous recurrence after H. pylori eradication of gastric neoplasms. Focusing on this mechanism may offer new insights into developing therapies to prevent gastric neoplasm recurrence after eradication. Currently, H. pylori eradication is the only treatment option for preventing metachronous recurrence of gastric cancer; however, our study suggests that patients who do not achieve immune recovery following eradication remain at risk for recurrence. These patients may benefit from adjunctive therapies aimed at restoring immune function, such as immune checkpoint inhibitors, which have shown potential in reactivating exhausted T‐cells and enhancing anti‐tumor immunity. Future studies with larger cohorts and prospective designs are needed to validate these findings and further elucidate the underlying mechanisms.
In conclusion, our study suggests that H. pylori eradication contributes to preventing metachronous recurrence in adenocarcinoma by improving histologic inflammation and enhancing immune system recovery. Specifically, the increase in CD8+ T‐cells and reduction in PD‐L1 expression post‐eradication could be critical factors in mitigating recurrence risk. These insights underscore the importance of immune modulation in cancer prevention and may guide future therapeutic strategies aimed at enhancing immune responses in patients with gastric cancer.
Author Contributions
Min‐Jae Kim: conceptualization: equal; data curation: equal; formal analysis: lead; investigation: equal; methodology: supporting; resources: lead; visualization: lead; writing – original draft: lead. Ji Hae Nahm: conceptualization: equal; data curation: equal; formal analysis: equal; investigation: lead; methodology: equal; visualization: equal; supervision: equal; writing – review and editing: equal. Yeonjin Je: conceptualization: equal; data curation: lead; formal analysis: supporting; funding acquisition: equal; investigation: equal; methodology: equal; resources: equal. Jaeyoung Chun: investigation: supporting; resources: supporting. Young Hoon Youn: investigation: supporting; resources: supporting. Hyojin Park: investigation: supporting; resources: supporting. Jie‐Hyun Kim: conceptualization: lead; funding acquisition: lead; methodology: lead; supervision: lead; writing – review and editing: lead.
Disclosure
The datasets generated and/or analyzed during the current study are not publicly available due to the policy of human‐derived materials research regulations.
Ethics Statement
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Yonsei University College of Medicine (IRB No. 3‐2023‐0325). The requirement for individual informed consent was waived due to the retrospective design of the study.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Table S1. Autoantibodies used in this study.
Acknowledgments
M.I.D. (Medical Illustration & Design), as a member of the Medical Research Support Services of Yonsei University College of Medicine, provides excellent support with medical illustration.
Funding: This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (2021R1A2C2011296). This study was supported by the Seyoung Association (SYH) research grant from the Department of Internal Medicine, Gangnam Severance Hospital (2022‐GNS‐004).
The first two authors contributed equally to this article.
The last two authors contributed equally to this article.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. Autoantibodies used in this study.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.