Performing the ABC Method Twice for Gastric Cancer Risk Stratification: A Retrospective Study Based on Data from a Large-Scale Screening Facility

The ABC method is a classification method used for stratifying the risk of gastric cancer. However, whether the ABC method should be performed only once or multiple times throughout an individual’s lifetime remains unclear. Therefore, this study aimed to analyze whether performing ABC screening twice in a lifetime is useful. We retrospectively analyzed the data of individuals who participated in health checkups in 2010 and 2015. We collected data on patient characteristics, pepsinogen levels, anti-Helicobacter pylori antibody titers, and the presence of gastric cancer. Overall, 7129 participants without a history of H. pylori eradication were included in this study. The participants’ average age in 2010 was 48.4 ± 8.3 years, and 58.1% were male. In addition, 11 and 20 cases of new H. pylori infection (0.15%) and spontaneous eradication (0.28%), respectively, were recorded. No significant difference was found in the incidence of gastric cancer between participants who underwent the ABC method once and those who underwent it twice (Group A: 0.16% vs. 0.16%; Group B: 0.47% vs. 0.39%; and Group C + D: 1.97% vs. 1.82%). Therefore, performing the ABC method twice, 5 years apart, does not significantly improve gastric cancer risk stratification.


Introduction
Helicobacter pylori (H. pylori) is a bacterial pathogen that infects more than half of the world's population [1]. It is believed to be transmitted from person to person through the oral-oral, gastro-oral, or fecal-oral routes, frequently during early childhood [2,3]. H. pylori infection is associated with chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue lymphomas, and gastric cancer.
The incidence of gastric cancer varies according to regions, with particularly high incidence rates in East Asia [4][5][6][7]. Upper gastrointestinal barium radiography and endoscopic gastric cancer screening are effective screening tests for gastric cancer. These tests allow for the early detection and treatment of gastric cancer, which can reduce mortality rates among patients [8]. Although upper gastrointestinal barium radiography and endoscopic gastric cancer screening are effective for detecting gastric cancer, they can be uncomfortable for patients and are associated with a small but significant risk of adverse events. In addition, the cost of these screening tests and the limited availability of endoscopists make it nearly impossible to screen a considerable proportion of the general population. Given these limitations, these tests should be reserved for high-risk cases. Therefore, effective methods for identifying high-risk groups, such as the measurements of pepsinogen (PG) level, anti-H. pylori antibody titer, and gastrin-17 level, are needed [9,10]. The ABC method, which combines the measurement of serum PG level and anti-H. pylori antibody titer, is an effective and convenient method of stratifying the risk for gastric cancer [11][12][13][14].
It has been reported that the ABC method is still useful for stratifying the risk of gastric cancer in Japan, even recently, where decreasing H. pylori infection rates have been reported [15]. Since new H. pylori infections in adults are rare, the ABC method is generally sufficient to be performed once in an individual's lifetime. However, there is no clear evidence whether the ABC method is sufficient once in a lifetime. As false-negative and false-positive results may be obtained and gastric mucosal atrophy may progress over time, some facilities perform the ABC method yearly.
High rates of reinfection after H. pylori eradication therapy have been reported in areas with high rates of H. pylori infection; however, the occurrence of new H. pylori infections in adults without a history of H. pylori eradication is unclear [16]. Recently, it was reported that H. pylori antibody titer and PG level change significantly in the early period after H. pylori eradication, albeit slowly [17]. However, how much atrophic gastritis has progressed over time in non-infected or currently infected patients in recent years is still unclear. In addition, no recent data exist on whether the measurement of anti-H. pylori antibody titer or PG level at different time points can affect the results of the ABC method.
Therefore, this study aimed to determine whether performing the ABC method twice is useful for gastric cancer risk stratification and to investigate the rates of new H. pylori infections and spontaneous H. pylori eradication in Japan using recent large-scale health checkup data.

Ethics Statements
The protocol for this study was approved by the Ethics Committees of the University of Tokyo (2865-(3)) and registered with UMIN-CTR (UMIN000013761). Written informed consent was obtained from each participant, and this study was performed in accordance with the principles of the Declaration of Helsinki.

Study Design and Participants
This was a retrospective cohort study conducted using the data of individuals who underwent health checkups at the Kameda Medical Center Makuhari (Chiba-shi, Chiba, Japan) in 2010 and 2015. Individuals who underwent the measurement of serum H. pylori antibody titer and PG I and II levels were included. However, individuals with a history of H. pylori eradication or those who did not undergo endoscopy or upper gastrointestinal barium radiography in 2010 or 2015 were excluded. Esophagogastroduodenoscopy (EGD) was recommended if abnormal findings were observed on upper gastrointestinal barium radiography. Data on age; sex; body mass index (BMI); anti-H. pylori antibody titer; pepsinogen level; smoking habits; use of proton pump inhibitors (PPIs), anticoagulants, nonsteroidal anti-inflammatory drugs (NSAIDs), or steroids; history of upper gastrointestinal tract surgery; the presence of gastric cancer; and history of H. pylori eradication in 2010 and 2015 were collected. Information on the history of H. pylori eradication, history of upper gastrointestinal tract surgery, smoking habits, and regular use of medications was confirmed using a questionnaire. Responses to the question on smoking habits were categorized into three, as follows: current, past, and never. Endoscopic data recorded up to December 2019 were referenced for determining the presence of gastric cancer.

Measurement of Anti-H. pylori Antibody Titer
The E-plate Eiken anti-H. pylori antibody kit (Eiken Chemical, Tokyo, Japan) was used the measure serum anti-H. pylori antibody titer in 2010, whereas the E-plate Eiken anti-H. pylori antibody II kit (Eiken Chemical Co., Ltd., Tokyo, Japan) was used in 2015.
Although the manufacturer's instructions state that the cut-off value for anti-H. pylori antibody titer is 10 U/mL, cases of H. pylori infection in patients with antibody titers of 3-10 U/mL are common [18,19]. Since April 2017, the cut-off value for the ABC method in Japan was adjusted to 3 U/mL to improve sensitivity [20]. As in previous studies, the cut-off value for negative H. pylori infection was set at <3 U/mL in this study. Anti-H. pylori antibody titers ranging from 3 U/mL to 9.9 U/mL were defined as "high-negative titers," whereas values ≥ 10 U/mL were considered positive. Cases in the high-negative titer group included current H. pylori infections and post-eradication cases. To reduce the incidence of false-positive results, new infections and spontaneous eradication of H. pylori were defined as cases where the patient's anti-H. pylori antibody titer changed from negative (<3 U/mL) to positive (≥10 U/mL), and those where the antibody titer changed from positive (≥10 U/mL) to negative (<3 U/mL) without H. pylori eradication therapy, respectively. The rates of new H. pylori infection and spontaneous H.pylori eradication were examined over the 5-year study period.

The ABC Method
LZ test EIKEN PG I and PG II (Eiken Chemical Co., Ltd.) were used to measure serum PG I and II levels. A PG test is considered positive if the PG I/II ratio and PG I are <3.0 and <70 ng/mL, respectively. In the ABC method, patients are categorized into four groups based on their PG and anti-H. pylori antibody test results, as follows: Group A, PG (−) and anti-H. pylori antibody (−); Group B, PG (−) and anti-H. pylori antibody (+); Group C, PG (+) and anti-H. pylori antibody (+); and Group D, PG (+) and anti-H. pylori antibody (−). We compared the risk of gastric cancer between participants classified using the ABC method in 2010 and those classified in both 2010 and 2015. Performing the ABC method twice was defined as assigning a participant to the same ABC group in 2010 and 2015, or to a higher group if the result obtained in 2015 was different (e.g., if the participant was assigned to Group B in 2010 and Group C in 2015, the participant's group would be Group C). The number of participants in Group D was small; therefore, it was combined with Group C for the analysis of the incidence rates of gastric cancer.

Statistical Analysis
All statistical analyses were performed using JMP 16.0 (SAS Institute, Cary, NC, USA) and Python 3.9 (Python Software Foundation, Wilmington, DE, USA). Categorical and continuous variables were analyzed using Pearson's chi-square or Fisher's exact tests and student's t-test, respectively. A two-sided p-value < 0.05 was considered statistically significant. Additional bootstrap tests with 1000 replications were performed to test for differences in the incidence rates of gastric cancer.

Results
A total of 9414 adults met the inclusion criteria. Of these, 2139 participants were excluded because they had a history of H. pylori eradication therapy. Of the remaining 7275 participants without a history of H. pylori eradication, 146 participants who had not undergone EGD or upper gastrointestinal barium radiography were excluded. Therefore, the remaining 7129 participants without a history of H. pylori eradication were analyzed. Subsequently, 6750 participants were analyzed after excluding cases of new H. pylori infections, spontaneous eradication, history of upper gastrointestinal tract surgery, and PPI use ( Figure 1). participants without a history of H. pylori eradication, 146 participants who had not dergone EGD or upper gastrointestinal barium radiography were excluded. Therefo the remaining 7129 participants without a history of H. pylori eradication were analyz Subsequently, 6750 participants were analyzed after excluding cases of new H. pylori fections, spontaneous eradication, history of upper gastrointestinal tract surgery, and P use ( Figure 1). The clinical characteristics of the participants are shown in Table 1. The participan average age in 2010 was 48.4 ± 8.3 years, and 4145 (58.1%) were male (Table 1). Overall, 11 and 20 cases of new infections (11/7129 (0.15%)) and spontaneous eradication (20/7129 (0.28%)), respectively, were recorded ( Table 2). Sex, age, BMI, history of upper gastrointestinal tract surgery, smoking, and use of PPIs, anticoagulants, steroids, or NSAIDs were analyzed to determine the odds ratios for the new H. pylori infection. For the new infections, the non-adjusted odds ratios for these variables were not significantly different. Age and use of anticoagulants showed significantly high non-adjusted odds ratios for spontaneous eradication. A multivariate regression analysis revealed that only age showed significant correlation to the spontaneous eradication of H. pylori (Table 3).
The When the participants whose ABC classifications in 2010 and 2015 did not match were classified into the higher-grade groups, Groups A, B, C, and D had 5543, 1036, 500, and 50 participants, respectively. The cancer incidence rate among participants who underwent the ABC method twice was 9/5543 (0.16%), 4/1036 (0.39%), and 10/550 (1.82%) for Groups A, B, and C + D, respectively. No significant difference was found in cancer incidence rates between participants who underwent the ABC method once and those who underwent it twice. In addition, the bootstrapping analysis results showed no significant differences in incidence rates (Table 4). To exclude factors that could affect anti-H. pylori antibody titer or pepsinogen level, a subgroup analysis was performed after excluding cases of new H. pylori infection, spontaneous H. pylori eradication, history of upper gastrointestinal tract surgery, or PPI use. After excluding 11 participants with new infections, 20 that showed spontaneous H. pylori eradication, and 348 with a history of upper gastrointestinal tract surgery or PPI use, the remaining 6750 participants were included in the analysis. Of the 6750 participants, 18 had gastric cancer. The cancer incidence rates among participants who underwent the ABC method in 2010 were 7/5356 (0.13%), 4/990 (0.40%), and 7/404 (1.73%) for Groups A, B, and C + D, respectively. A total of 313 participants were classified into different groups based on whether they underwent the ABC method once or twice. When participants whose classifications in 2010 and 2015 did not match were classified into the higher grade groups, Groups A, B, C, and D had 5302, 957, 458, and 33 participants, respectively. The cancer incidence rates among participants who underwent the ABC method twice were 7/5302 (0.13%), 3/957 (0.94%), and 8/491 (1.63%) for Groups A, B, and C + D, respectively. No significant difference was found in the cancer incidence rate between participants who underwent the ABC method once and those who underwent it twice. The bootstrapping analysis results showed no significant differences in incidence rates as well (Table 5).

Discussion
In this study, we used large-scale health checkup data to determine whether it is useful to perform the ABC method twice and to examine the frequency of new H. pylori infections and spontaneous H. pylori eradication in adults. The results showed that there was no significant difference in the incidence rate of gastric cancer between the participants who underwent the ABC method once and those who underwent it twice. It appears that there is no need to repeat the ABC method in less than 5 years. In addition, 11 new H. pylori infections (11/7129 = 0.15%) and 20 spontaneous H. pylori eradication (20/7129 = 0.28%) were recorded within the 5-year study period. New H. pylori infection and spontaneous H. pylori eradication rates in adults were estimated to be 0.03% and 0.06% per person-year, respectively, in this study. These rates are lower than those reported in previous studies, where seroconversion (positive to a negative change in anti-H. pylori Immunoglobulin G (IgG) level and seroreversion (negative to a positive change in anti-H. pylori IgG level) rates were reported to be 0.53-0.70% and 0.11-24% per person-year, respectively [21][22][23]. In this study, new H. pylori infection was defined as an increase in anti-H. pylori antibody titer from <3 U/mL to ≥10 U/mL, whereas spontaneous H. pylori eradication was defined as a decrease in anti-H. pylori antibody titer from ≥10 U/mL to <3 U/mL. Compared with previous studies, cases in this study where anti-H. pylori antibody titers increased from 3-9.9 U/mL to ≥10 U/mL were uncategorized as new H. pylori infections. If new H. pylori infections in this study were defined as an increase in anti-H. pylori antibody titer from <10 U/mL to ≥10 U/mL as in previous studies, the rate of new H. pylori infection in this study would be 0.50% (36/7129). Similarly, if spontaneous H. pylori eradication was defined as a decrease in anti-H. pylori antibody titer from ≥10 U/mL to <10 U/mL, the rate of spontaneous H. pylori eradication in this study would be 1.3% (93/7129). Although these results were excluded from the main results, they are comparable to those of previous studies. In Japan, the prevalence of H. pylori has been decreasing because of improved sanitary conditions [24]. Recently, the prevalence of H. pylori in children aged 0-8 years was reported to be 1.9% without new H. pylori infections recorded during a 1-year follow-up period [25]. As the rate of H. pylori infection continues to decrease, the rate of new H. pylori infections is also expected to decline.
Miki et al. suggested that the ABC method can be used for national mass stratification for gastric cancer risk [11]. The effectiveness of the ABC method has been reported; however, the effectiveness of multiple ABC methods has not. In this study, we analyzed the incidence of gastric cancer at the end of 2019 in participants who underwent the ABC method in both 2010 and 2015. Even after excluding cases of new H. pylori infections, spontaneous H. pylori eradication, history of upper gastrointestinal tract surgery, and PPI use, no significant difference was found in cancer rates between participants who underwent the ABC method once and those who underwent it twice. These results were confirmed using a sufficiently large number of cases, and we considered them reasonable because they did not change after the bootstrapping analysis was performed.
Pepsinogen level is reported to barely fluctuate within approximately 10 years in more than 90% of adults [11]. It is possible that this stability in the pepsinogen level may be responsible for the lack of significant differences between participants who underwent the ABC method once and those who underwent it twice in Japan, where the rate of new H. pylori infection is low. Although assessment using the ABC method every 5 years did not improve gastric cancer risk stratification, it allowed for the identification of some cases of new H. pylori infections.
In this study, no participants with new H. pylori infection developed gastric cancer. The long-term risk of developing gastric cancer in adults with new H. pylori infections remains unknown. Therefore, further studies at long intervals are needed to clarify if the ABC method is sufficient once in a lifetime. The ABC method is used for stratifying the risk of gastric cancer in patients without a history of H. pylori eradication. As more people are eradicated of H. pylori, new screening methods for patients with a history of H. pylori eradication should be considered.
Spontaneous eradication of H. pylori was more likely to occur in the older adults and anticoagulant users in this study. Subjects with anticoagulants use were significantly older than those without anticoagulants use. The mean age of the 153 subjects with anticoagulants use was 57.7(±9.5) years, and the mean age of the 6976 subjects without anticoagulants use was 48.1 (±8.1) years. (p < 0.01) There is a correlation between age and anticoagulant use, and the multivariate regression analysis revealed that only age showed significant correlation to spontaneous eradication. The success rate of H. pylori eradication is higher in older adults than in the younger adults, and it has been suggested that decreased gastric acid capacity may be involved in this [26]. It is possible that the use of antibiotics for other reasons combined with the low gastric acidity may have caused an unintended eradication of H. pylori.
This study had some limitations. First, different reagents were used for measuring anti-H. pylori antibody titer in 2010 and 2015. Although we cannot exclude the possibility that the results may have been affected by this use of different reagents, we believe that the effect was minimal because of the high concordance rates of the anti-H. pylori antibody titers recorded. Second, definitions of new H. pylori infection and spontaneous H. pylori eradication were solely based on anti-H. pylori antibody titer, which may result in falsepositive results. It has been reported that PGI and II are increased and PGI/PGII is decreased in patients with H. pylori infection, and that PGI and PGII are decreased and PGI/PGII is increased after the eradication of H. pylori. [27,28] In this study, the PG I and II levels of the 11 participants with new H. pylori infections tended to increase after infection; however, the difference in PG levels was not significant. In addition, the results indicated that PG I/PG II significantly decreased after infection (PG I:  Third, not all participants were followed up until the end of 2019. In addition, EGD or upper gastrointestinal barium radiography is selected based on patient preference and gastric cancer screening methods are not standardized. However, 5222 of 7129 individuals (73.3%) were followed up until 2019 using upper gastrointestinal barium radiography or EGD. We believe that the number of participants who were followed up is sufficient to draw valid conclusions from this study's results. Finally, we used a questionnaire to determine whether a participant had received H. pylori eradication therapy and whether it was successful. However, it should be noted that the evaluation of H. pylori eradication history in the general population using a questionnaire is considered reliable and valid [29].
In conclusion, this study demonstrated that performing the ABC method twice, 5 years apart, does not significantly improve gastric cancer risk stratification. In addition, this study showed that in Japan, the rates of new H. pylori infection and spontaneous H. pylori eradication over 5 years were 0.15% and 0.28%, respectively.  Institutional Review Board Statement: The protocol for this study was approved by the ethics committees of the University of Tokyo (2865) and registered with UMIN-CTR (UMIN000013761).

Informed Consent Statement: Written informed consent was obtained from each participant.
Data Availability Statement: The data are not publicly available due to the decision of the Ethics Review Committee.