Clinicopathological Characteristics and Endoscopic Features of Early Gastric Cancers Diagnosed After Helicobacter pylori Eradication: A Retrospective Study

Background: Helicobacter pylori (H. pylori) infection is an important risk factor for developing gastric cancer. However, even after H. pylori eradication, early gastric cancer (EGC) can develop. We elucidated the characteristics of EGCs diagnosed after H. pylori eradication. Methods: Thirty-six EGCs in 32 patients diagnosed after H. pylori eradication were dened as the eradication group (H. pylori-EG). The clinicopathological and endoscopic features were compared with those of 156 EGCs in 140 patients in the H. pylori-positive group (H. pylori-PG). Twenty-nine EGC lesions in the H. pylori-EG were further divided into two subgroups: the rst included six lesions of none to mild atrophic mucosa around the EGC, and the second included 23 lesions of moderate to severe atrophic mucosa around the EGC. We compared them between the two subgroups. Results: Endoscopic features of EGCs in the H. pylori-EG were characterized as small (P = 0.049) and of the depressed type (P = 0.022) compared with those in the H. pylori-PG. EGCs in the H. pylori-EG were detected on the upper region of the stomach more frequently than those in the H. pylori-PG (P = 0.002). As for submucosal ECGs in the H. pylori-EG, it was more likely to be seen in the mild atrophic mucosa subgroup (4/6, 67%) compared to the moderate to severe atrophic gastric mucosa subgroup (1/23, 4%) (P = 0.003). Conclusions: EGCs after H. pylori eradication were characterized as small and of the depressed type. Submucosal invasive EGCs developed more frequently in the none to mild atrophic mucosa after H. pylori eradication. Therefore, careful patient follow-up is important after H. pylori eradication.


Background
The infection with Helicobacter pylori (H. pylori) was classi ed as a carcinogen of gastric cancer by the World Health Organization in 1994. In patients with early gastric cancer (EGC) who underwent endoscopic resection, EGCs were detected in H. pylori-positive patients at a signi cantly higher rate than in H. pylorieradiation patients [1]. In a multicenter randomized controlled trial in Japan, the incidence of metachronous cancer was 14.1 cases per 1,000 person-years in the H. pylori-eradication group and 40.5 cases per 1,000 person-years in the no eradication group (hazard ratio 0.339, 95% CI 0.157-0.729, P = 0.003) [2]. The effectiveness of H. pylori eradication was proved by the decrease in the incidence of gastric cancer.
Because there are only several reports concerning gastric cancer after H. pylori eradication, we conducted a retrospective study investigating the clinicopathological characteristics and endoscopic features of EGCs diagnosed after H. pylori eradication.

Study Subjects
The study was conducted at Fukuoka University from January 2009 to January 2018. The Medical Ethics Committee of Fukuoka University approved this study (IRB number: U20-01-011).

Primary Endpoint
Primary endpoint of this study to investigate the association between the clinicopathological characteristics and endoscopic features of EGCs diagnosed after H. pylori eradication. We enrolled 397 consecutive patients who underwent endoscopic submucosal dissection (ESD) treatment of their EGC. Of these, 225 patients were excluded as follows: 89 patients were not examined for H. pylori status; in 124 patients, H. pylori was not detected, and these patients did not have a past history of receiving H. pylori eradication therapy; and in another 12 patients, EGCs were resected using ESD within 1 year after H. pylori eradication. The remaining 172 patients were divided into two groups: An H. pylori-eradication group (H. pylori-EG) that comprised 36 EGCs in 32 patients who had undergone H. pylori eradication therapy more than 1 year before and on whom ESD was performed to treat their EGC, and an H. pyloripositive group (H. pylori-PG; the control group) that comprised 156 EGCs in 140 patients with an active H. pylori infection and on whom ESD was performed to treat their EGC (Fig. 1).

Secondary Endpoint
It remains unclear whether the mucosal atrophy affect the characteristics of EGC after eradication of H. pylori. Therefore, we compared the clinicopathological characteristics and endoscopic features between the two groups classi ed by the degree of gastric mucosal atrophy in the H. pylori-EG group. To investigate the association between EGC characteristics and the degree of mucosal atrophy after H. pylori eradication, 29 lesions of EGC in the H. pylori-EG group, in which the degree of mucosal atrophy had been con rmed in ESD specimens, were divided into two subgroups according to endoscopic and histological examinations. The mild atrophic mucosa subgroup comprised 6 lesions that were none to mild atrophic mucosa around the EGC, and the moderate to severe atrophic mucosa subgroup comprised 23 lesions that were moderate to severe atrophic mucosa around the EGC (Fig. 1).

Evaluation of H. pylori status
Evaluation of H. pylori eradication treatment was based on the 13 C-urea breath test (UBT) or serum immunoglobulin (Ig) G antibody test (E-plate, Eiken, Tokyo, Japan) and on histological assessment using endoscopic biopsy specimens. When both examinations were negative, we determined that H. pylori had been eradicated. One hundred and forty patients in the control group were H. pylori-positive based on UBT or serum IgG antibody and histological assessment, and had no history of receiving H. pylori eradication therapy.

Clinicopathological Assessment
Clinicopathological ndings such as size, location, macroscopic type, histological type, and depth of tumor invasion were reviewed for gastric carcinomas according to both the Japanese [8] and World Health Organization classi cation [9]. The extent and degree of atrophic gastritis was evaluated endoscopically and histologically and classi ed into six categories according to the Kimura and Takemoto classi cation system (C-1 to O-3) [10].

Endoscopic Procedures
Endoscopic examinations were performed by three experienced endoscopists (H.I, T.T, N.K) using a magnifying endoscope (GIF-H260Z, H290Z, Olympus Medical System, Tokyo, Japan). We used structural enhancement levels of A-8 for conventional endoscopy and B-8 for narrow-band imaging with magnifying endoscopy (NBI-ME). NBI-ME for diagnosing EGC was performed using a systematic classi cation system based on microvascular patterns and microsurface patterns (the "VS classi cation"). An irregular microvascular pattern (IMVP) and/or an irregular microsurface pattern (IMSP) with clear demarcation lines are the hallmarks of EGC [11]. EGCs were con rmed using histopathological ndings of biopsies and ESD samples.

Statistical Analysis
Data were analyzed using JMP ® 15 statistical software (SAS Institute Inc., Cary, NC, USA). Continuous variables between two groups were evaluated using the Mann-Whitney test. Categorical variables were evaluated using the chi-squared test. P values of <0.05 were considered statistically signi cant.

Results
Clinicopathological characteristics and endoscopic features of early gastric cancers of the H. pylorieradication group and the H. pylori-positive group Clinicopathological characteristics and endoscopic features EGCs of the H. pylori-EG and the H. pylori-PG are summarized in Table 1. There were 36 EGCs in 32 patients in the H. pylori-EG and 156 EGCs in 140 patients in the H. pylori-PG. The median patient age in the H. pylori-EG (68.1±7.3) was signi cantly younger than that of patients in the H. pylori-PG (71.5±8.7) (P = 0.038). The median lesion size in the H. pylori-EG (12.7±8.2 mm) was signi cantly smaller than that in the H. pylori-PG (16.5±10.8 mm) (P = 0.049). EGCs were frequently detected on the upper region of the stomach in six of 36 patients in the H. pylori-EG (17%) compared with ve of 156 in the H. pylori-PG (3%; P = 0.002). Regarding macroscopic features, elevated lesions were more frequent in 57 of 156 patients in the H. pylori-PG (36%) compared with six of 36 in the H. pylori-EG (17%; P = 0.022). Depressed lesions were more frequent in 30 of 36 patients in the H. pylori-EG (83%) compared with 99 of 156 in the H. pylori-PG (64%; P = 0.022). Close type-3 in extent of atrophic gastritis according to Kimura and Takemoto classi cation system [10] was more frequent in the H. pylori-EG (8/36: 22%) compared with the H. pylori-PG (9/156: 6%) (P = 0.0002). Open type-1 was more frequent in the H. pylori-PG (67/156: 43%) compared with the H. pylori-EG (6/36: 17%) (P = 0.0002). EGCs after H. pylori eradication were characterized by features of the small and depressed type, and frequently detected on the upper region of the stomach compared with EGCs in the H. pylori-PG. In histological ndings such as histologic type, depth of invasion, and lymphatic and venous invasion, there were no differences between the two groups.
Association between characteristics of early gastric cancers and the degree of mucosal atrophy in the H. pylori-eradication group Clinicopathological characteristics and endoscopic features of EGCs of the mild atrophic gastric mucosa subgroup and the moderate to severe atrophic gastric mucosa subgroup within the H. pylori-EG are summarized in Table 2. Table 2 shows that six lesions (21%) were in the mild atrophic mucosa subgroup and 23 lesions (79%) were in the moderate to severe atrophic mucosa subgroup. Three of 6 mild atrophic subgroup (50%) had none atrophic mucosa around EGC. There were no signi cant differences in age, interval term from the eradication of H. pylori to the detection of the EGC, location, size, macroscopic nding, histologic type, or lymphatic and venous invasion between the two subgroups. In the moderate to severe atrophic subgroup, there were more males than in the mild atrophic mucosa subgroup (P = 0.02). Regarding depth of invasion, submucosal invasive EGC lesions were detected in four of six lesions (67%) in the mild atrophic mucosa subgroup and only 1 of 23 lesions (4%) in the severe atrophic mucosa subgroup (P = 0.003). Three of 4 submucosal invasive EGC lesions (75%) in mild atrophic subgroup had none atrophic mucosa around EGC.

Median interval from the eradication of H. pylori to the detection of EGC
In the H. pylori-EG, EGCs were detected 1-17 years (median, 4.6 years) following H. pylori eradication. In the mild atrophic gastric mucosa subgroup within the H. pylori-EG, the median interval from eradication therapy to detection of an EGC was 5.3 years (1-17 years). In the moderate to severe atrophic gastric mucosa subgroup, the median interval was 4.3 years (1-17 years). Median intervals were not signi cantly different between the two subgroups (Fig. 2). This result showed that there was no signi cant association between the duration of EGC incidence and the degree of mucosal atrophy.

Submucosal invasive early gastric cancer in the H. pylori-EG group
The clinicopathological characteristics and endoscopic features of submucosal invasive ve EGCs in the mild atrophic gastric mucosa subgroup and moderate to severe atrophic mucosa subgroup within the H. pylori-EG are summarized in Table 3. Endoscopic features of EGCs in all cases showed a attened and extended appearance when the entire stomach wall was distended with a high volume of air. Therefore, patients were diagnosed with intramucosal gastric cancer because they were negative for the nonextension sign [12]. Three cases (case1, 3, 4) had none atrophic mucosa around EGC. Endoscopic and pathological ndings of cases 1 and 4 are shown in Figures 3 and 4, respectively. In case 1, upper gastrointestinal (GI) endoscopy revealed a reddish small elevated lesion on the upper position of the gastric cardia (Fig. 3a). NBI-ME revealed a regular microsurface pattern and an absent microvascular pattern (Fig. 3b). The tumor was successfully removed en bloc using the ESD method (Fig. 3c). The resected specimen was evaluated histopathologically and revealed a well differentiated tubular adenocarcinoma invading into the submucosa (SM1) (Fig. 3d), none atrophic mucosa around EGC (Fig.  3e). In case 4, upper GI endoscopy revealed a reddish small depressed lesion on the upper position of the gastric cardia (Fig. 4a, b). The tumor was successfully removed en bloc using the ESD method (Fig. 4c). Histopathological examination revealed a well-differentiated tubular adenocarcinoma invading into the submucosa (SM2) and none atrophic mucosa around EGC (Fig. 4d).

Discussion
Because a meta-analysis of clinical studies indicated that gastric cancers occurred even after H. pylori eradication [4,13,14], it is important to recognize the clinical and histological characteristics of gastric cancers detected after H. pylori eradication for making the appropriate diagnosis and treatment. A previous report described 16 gastric cancers detected after H. pylori eradication that were characterized by a noncardiac location and less than 20 mm in size with depressed type features [15]. Furthermore, 96 gastric cancers detected after H. pylori eradication had endoscopic features of a depressed type and were small in size compared with H. pylori-positive cancers [4]. Gastric cancers detected after H. pylori eradication were characterized by a small size, a depressed type, and a lower Ki-67 labeling index compared with H. pylori-positive gastric cancers [3]. Likewise, the H. pylori-EG had more frequent small lesions and depressed types compared with the H. pylori-PG (P = 0.049, P = 0.022) in our study. The current study also showed that the features of EGC after H. pylori eradication were characterized as small lesions of the depressed type.
The prevalence of gastric cancers located on the upper region of the stomach was higher in the eradication group compared with the no eradication group, which suggests that gastric cancer developed on the upper region of the stomach after H. pylori eradication [5]. In our study showed the same tendency, EGCs were detected more frequently on the upper region of the stomach in the H. pylori-EG compared with the H. pylori-PG (P = 0.002). Hence, it is necessary to perform careful examination of the upper region of the stomach using upper GI endoscopy after treating a patient with H. pylori eradication therapy.
Several studies showed that patients with gastric cancer detected after eradication of H. pylori had severe atrophic gastritis [15,16]. Atrophy of gastric mucosa in the antrum, angle, and corpus, and intestinal metaplasia in the lesser curvature of the corpus showed signi cant improvement during the 10-year period after eradication therapy. Therefore, the improvement of gastric atrophy and intestinal metaplasia were associated with a reduction in gastric cancer occurrence [17]. However, the risk of developing a diffuse-type gastric cancer increases over time in patients with mild to moderate gastric atrophy before H. pylori eradication [18]. In this study, six of 29 cases (21%) were none to mild atrophic gastritis around the gastric cancer that occurred after H. pylori eradication. Our result strongly suggested that there was an incidence risk of EGC even in patients with none to mild atrophic gastritis after H. pylori eradication, although the incidence risk of EGC in patients with none to mild atrophic gastritis was lower than in those with severe atrophic gastritis.
Recently, several studies reported that there were cases of submucosal invasive EGCs after H. pylori eradication. In 162 patients with H. pylori eradication and non-H. pylori eradication, submucosal invasive EGCs were detected more frequently in the H. pylori-eradication group (13/81 patients, 16%) than in the non-H. pylori-eradication group (4/81 patients, 4.9%; P = 0.021) [19]. The submucosal invasive gastric cancer tended to be more frequent in the H. pylori-EG (18%) than in the non-H. pylori-EG (8%; P = 0.051) [4]. We described four cases of submucosal invasive EGCs with none to mild atrophic gastric mucosa after H. pylori eradication therapy. They were endoscopically diagnosed as intramucosal gastric cancer because of a negative non-extension sign as shown in case presentation [12]. Despite none to mild atrophy after H. pylori eradication, it is necessary to follow up using GI endoscopy because of the possibility of EGC and the high risk of invasive cancer.
A 9-year prospective study in Japan showed that 20 (1.1%) gastric cancers were diagnosed in 1,787 patients who underwent H. pylori eradication therapy. Of these 20 gastric cancers, 16 (80%) were diagnosed within 4 years after H. pylori eradication [15]. Previous studies reported that the median intervals from H. pylori eradication therapy to detection of gastric cancers were 2.6 years (range 0.8-11 years) [3], 4.1 years (1-15 years) [4], and 3 years (0.5-15 years) [5]. In this study, the median interval was 4.6 years (1-17 years). In the mild atrophic gastric mucosa subgroup of the H. pylori-EG, the median interval was 5.3 years (1-17 years). In the moderate to severe atrophic gastric mucosa subgroup, the median interval was 4.3 years (1-17 years). We suggested that upper GI endoscopic examination should be performed in patients in whom H. pylori had been eradicated for at least 6 years, regardless of the degree of atrophic mucosa. However, few cases of EGC were detected 15 or 17 years after H. pylori eradication therapy. Endoscopic surveillance should be continued beyond 10 years after H. pylori eradication regardless of the degree of gastric mucosal atrophy [18].

Conclusions
Our single-center study revealed that EGCs after H. pylori eradication were characterized endoscopically with features of small and depressed lesions and invasive carcinogenesis in the none to mild atrophic mucosa. Therefore, we should perform careful follow-up with upper GI endoscopic examination after H. pylori eradication, with special attention to small and depressed type gastric cancer for at least six years, but not more than 15 or 20 years. This study had several limitations. The study was retrospective and conducted at a single center. Because there are few reports that evaluate EGCs after H. pylori eradication, it will be necessary to perform a prospective study across multiple patient populations and ethnic groups in the future. Abbreviations H. pylori: Helicobacter pylori, EGC: Early gastric cancer, ESD: Endoscopic submucosal dissection UBT: 13 C-Urea breath test, NBI-ME: Narrow-band imaging with magnifying endoscopy IMVP: Irregular microvascular pattern, IMSP: Irregular microsurface pattern, GI: Gastrointestinal Declarations Ethics approval and consent to participate This study conducted in accordance with the ethical standards of the declaration of Helsinki. This study protocol was approved by the Human Ethics Review Committee of Fukuoka University, Japan (IRB number: U20-01-011). Informed consent to participate this study was obtained from all patients in orally. The procedure for oral consent was approved by the ethics committee.

Consent for publication
Not applicable.

Availability of date and materials
The datasets used and analyzed in the current study are available from the corresponding author on reasonable request.

Competing interests
The author declare that they have no competing interests.

Funding
This study received no nancial support.
Author's contributions HI and FH designed the research. HI, TT, SI, HM, HY, HS, SM, YK, NK, KA, SF performed research. HI, TT performed statistical analysis. HI, HT and FH wrote the paper. All authors read and approved the nal manuscript.    Figure 1 Flow of the study subjects.