Relationship between Helicobacter Pylori Infection and the Risk of Esophageal Cancer in Thailand

Objective: Esophageal cancer (EC) is a multifactorial disease and a leading cause of mortality. Epidemiological and molecular studies have provided evidence that Helicobacter pylori (H. pylori) infection is an important cause of gastric carcinogenesis and thus, may be related to EC. However, esophagus H. pylori infection in Thai patients with newly diagnosed EC has not been reported. Moreover, the evidence of the association with H. pylori to EC is controversial. This study investigated the possible association between H. pylori infection with a virulence gene and EC in Thailand. Methods: A case-control study was conducted that involved 105 newly diagnosed EC patients and 108 healthy controls. The prevalence of H. pylori infection detected in formalin-fixed, paraffin-embedded EC tissue in esophageal biopsy specimens from the subjects was measured using real-time PCR. All the data were collected in face to face interviews using a structured questionnaire. Multivariable unconditional logistic regression was used to calculate and analyses the odds ratios (ORs) of the data. Results: A significant association was found between H. pylori infection and EC (p < 0.001, 95% CI:3.11–10.48). H. pylori-positive subjects had a 2.76 times higher risk of developing ESCC. Moreover, the H. pylori-positive subjects who were CagA-positive had slightly higher ORs and statistically significant risk factors. Conclusions: H. pylori infection was found to be associated with a risk of EC in Thailand, and among the H. pylori-positive subjects who were CagA-positive had a higher risk factor of ESCC but not of EAC.


Introduction
Esophageal cancer (EC) represents a major problem globally. It is one of the most frequently occurring malignancies in the world, and its incidence and mortality are increasing (Ferlay et al., 2019). There are two major histological subtypes of EC: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC and EAC have different demographic and geographic models (Gao et al., 2019). In Thailand, EC is a chronic disease, a serious health problem and the eighth leading cause of death. Its overall estimated age standardized incidence rates among men and women in 2015 are 4.8 and 1.4 cases per 100,000, respectively. Moreover, the number of new cases increase every year (Imsamran W et al., 2015). Many studies have confirmed the role of tobacco smoking, alcohol drinking and poor oral hygiene in the etiology of EC (Chen et al., 2017;Geßner et al., 2021;Jayalekshmi et al., 2021). Some studies have found, however, that Helicobacter pylori (H. pylori), an infectious agent that is known to be an important risk H. pylori infection and some diseases, but that only 20% of H. pylori-infected patients develop a serious disease (Sonnenberg, 2022). The extent and the severity of these associated diseases depend on several factors, such as the bacterial virulence factors and the host factors such as the immune system and environmental determinants (Amieva et al., 2016;Yamaoka et al., 2014). H. pylori has a number of virulence factors that play a role in its pathogenesis and that influence its colonization, invasion and disease severity (Maleki Kakelar et al., 2019;Raghwan et al., 2014). Previous studies demonstrated that the virulence genes of H. pylori play an important role in its pathogenicity (Baj et al., 2020). The most widely studied H. pylori virulence factor is the cytotoxin-associated gene A (CagA) antigen, a 96-138-kDa protein. The CagA gene was found in the genomic region called the Cag pathogenicity island (PAI), and it is considered a marker of an enhanced virulence gene. H. pylori strains that are CagA-positive are associated with severe inflammation and increased risk of ulcers and cancer in humans (Noto et al., 2012;Palrasu et al., 2020;Yamaoka et al., 2014). Various studies have been conducted to demonstrate the relationship between virulence genes of H. pylori and the severity of gastrointestinal diseases, and most of those studies investigated CagA genes (Abu-Taleb et al., 2018). Several epidemiological studies have examined the association between H. pylori infection, including infection with the more virulent CagA-positive strains, and the risk of EC (Anderson et al., 2008). However, such studies have reported inconclusive results, and some of them even found a reverse relationship between H. pylori and ESCC after performing a serological test or ELISA (Xie et al., 2013). Similar studies have presented some evidence, following polymerase chain reaction (PCR), that CagA-positive H. pylori infection is associated with the risk of EC or gastric cancer (Glocker et al., 1998;Krashias et al., 2013;Li et al., 2014). In Thailand, the prevalence of H. pylori virulence gene infection in the upper aerodigestive tract (UADT), which includes the esophagus, is unknown in the general population but only in patients with EC undergoing upper endoscopy, and no study has simultaneously examined the relationship between the H. pylori CagA virulence gene and EC patients. Therefore, the main objective of this study is to investigate the association between H. pylori CagA gene infection and EC in patients in a hospital through a case-control study to inform the planning of a large-scale etiological study of UADT cancer in Thailand.

Study design and sample collection
This study is a hospital-based case-control study that involved 105 patients with EC and 108 healthy controls recruited from Srinagaring Hospital in Khon Kaen province, northeast Thailand, from 2007 to 2017. Case subjects were defined as patients who had a histopathologically confirmed diagnosis of EC. Control subjects were patients with a diagnosis of non-cancer condition. They were selected from patients undergoing routine endoscopy for investigation of presumed nonmalignant conditions such as gastroesophageal reflux disease. All the patients were admitted at the same time and in the same ward as cancer cases. Pathological physicians diagnosed the inclusion criteria in all the cases. The data on risk factors were collected by a trained interviewer using a standardized questionnaire developed by researchers. EC specialists confirmed the validity of the content of the questionnaire. All the subjects were asked to answer, face to face, another questionnaire on their demographic characteristics and the history of their illness. Tissue samples were collected from all the subjects. The specimen tissue was separated into two groups: EC cases and Control subjects. ESCC and EAC tissues were included in the EC cases group, and normal esophageal tissues were put in the Control subjects group. All these 213 retrospective formalin-fixed, paraffinembedded (FFPE) tissue samples, which included 105 EC and 108 normal tissues, were retrieved from paraffin blocks stored at Khon Kean University (Department of Pathology, Faculty of Medicine) from 2007 to 2017. They were initially diagnosed by a specialist according to the International Classification of Diseases for Oncology Third Edition and confirmed via electronic gastroscopy and histopathology reports. This study was approved by the Khon Kaen University Ethics Committee in Human Research (reference no. HE621269).

DNA extraction
The total genomic DNA were extracted from the FFPE esophagus tissue using the commercially available DNeasy blood and tissue kits (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The DNA quality and concentration were quantified with a Nano Drop ND-1000 spectrophotometer (Thermo Scientific). All the DNA samples were stored at -20°C for further analyses.

H. pylori detection via SYBR Green based real-time PCR assay
After the DNA sample extraction, PCR was performed to detect H. pylori using specific primers, target genes, amplicon sizes, sequences and primer names. All the primers used in this study are presented in Supplementary Table 1. Real-time PCR assays were performed on an Applied Biosystems 7,500 flats system (Applied Biosystems, USA) using the SYBR Green PCR kit (Fermentous, Germany) to confirm the presence of H. pylori CagA-positive DNA samples. For PCR amplification, 1-2 μg of DNA samples was added to a PCR mixture that contained 12.5 μl of 2x SYBR Green mastermix, 0.3 µM each of the forward and reverse primers, and sterile water to achieve a total volume of 25 μl. PCR amplification was performed under the following conditions: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing for 30 s, primer extension at 72°C for 30 s and final extension at 72°C for 5 min. The data were analysed using QuantstudioTM 6 Flex Real-Time PCR systems. The H. pylori strain was detected using specific primers that targeted 16S rRNA and ureA genes. The CagA-positive status was determined from H. pylori-positive samples via PCR using their respective primers, as described in

Demographic characteristics
The characteristics of the 105 patients with EC and the 108 normal controls are described in Table 1. The mean age (± SD) at diagnosis was 60.4 years (± 8.9), and 63 (60%) of the included samples were from male patients. For the demographics of the subjects' lifestyle habits, drinking, smoking and daily proton pump inhibitor (PPI) use were more prevalent among the EC cases than the controls. The frequency of a personal history of gastroesophageal reflux disease (GERD) differed between the cases and the controls (p-value = ≤ .05). There was a higher prevalence of H. pylori-positive specimens (79%) and CagA-positive specimens (24%) among the EC cases than among the controls (38% and 9.3%, respectively). Table 2 presents the factors associated with EC and the main outcomes of the multivariable analysis. The factors found to be significantly associated with EC are alcohol consumption, smoking and a personal history of GERD. There was a statistically significant dose-response relationship (p-value < .05). We also found that among the sample genes (ureA and 16S rRNA), 123 (57%) were H. pylori-positive. These included 83 (79%) among the EC cases and 40 (38%) among the controls. Infection Supplementary Table 1. Both a positive control (DNA extracted from reference stains, the 16S RNA genes of which were sequenced) and a negative control (molecular distilled water) were used in each run of the PCR assay.

Statistical analysis
The statistical analysis was performed with STATA software, version 13.0. The proportions of the specimens that tested positive for H. pylori (overall, if either the ureA genes or the 16S rRNA genes were amplified) were expressed as percentages. Among the H. pyloripositive specimens, the proportion that tested positive for the virulence gene CagA were also calculated. Bivariate analysis using simple logistic regression was performed to investigate the association between the independent factors and EC, without controlling for variable confounding. Crude odds ratios (OR crude) and their 95% confidence intervals (95% CI) were determined. Multivariable unconditional logistic regression was used to compute adjusted odds ratios (OR adj ) and their 95% CI to investigate the association between H. pylori infection, CagA gene infection and EC while controlling for the effects of confounding variables. All the test statistics were two-sided, and a p-value of ≤ .05 was considered statistically significant.

Discussion
To our knowledge, current data show that smoking, drinking alcohol, and a personal history of GERD and H. pylori infection are all associated with an increased risk of EC and are the major etiology of EC in the Thai population. Interestingly, these important data also found an association between H. pylori infection and an increased risk of ESCC. Several studies have focused on the diversity of H. pylori infection and its virulence genes in different countries (Ofori et al., 2019;Subsomwong et al., 2017). Whereas a previous systematic review has reported that the association of H. pylori infection with EC was controversial, because almost all the studies have confirmed of H. pylori infection using a serological test, the test might have involved overlapping cases of esophageal and gastric infections (Xie et al., 2013). Two studies confirmed that esophageal infection with CagApositive H. pylori, which was detected via PCR, was associated with ESCC (Krashias et al., 2013;Li et al., 2014). However, there is little information available related to the frequency of H. pylori CagA infection among EC patients in Thailand, despite the fact that CagA is the protein necessary for NF-κB activation, which results in IL-8 secretion, neutrophil accumulation and tissue necrosis (Glocker et al., 1998) . Therefore, we conducted a case-control study to detect the H. pylori CagA gene DNA in the esophagus tissue of EC patients and normal controls using real-time PCR. Our study investigated the relation of the CagA virulence gene in H. pylori infection and if it is associated with the incidence of EC. In this study, we observed a high prevalence (57.7%) of H. pylori infection in the esophagus tissues of our 213 subjects. Similarly, several studies found from a nationwide survey in Thailand that among 1,546 patients from 17 provinces in four regions, the overall prevalence of H. pylori infection was 55.9% (Uchida et al., 2015), and some s t u d i e s b a s e d o n c u l t u r e a n d h i s t o l o g y immunohistochemistry found that the prevalence of H. pylori infection was 54.5% in North Thailand (Subsomwong et al., 2017). These findings are consistent with those of other studies (Tunruttanakul S et al., 2018). However, studies from other countries in the same region, such as Vietnam and Malaysia, have shown low H. pylori prevalence, epidemiologically diverse H. pylori infections, and different prevalence rates in different geographical locations (Fock et al., 2010). An important finding is that H. pylori positivity was associated with EC in Thailand. In this study that used archived FFPE tissues of EC patients diagnosed in the years 2007-2017, H. pylori DNA was detected in 79.1% of the tissues, which demonstrated that H. pylori infection was associated with a progression of EC. This result revealed that H. pylori infection is associated with a statistically significant increased EC risk (Odd adj , 3.8; 95% CI, 3.11-10.48). These data also reported an odds ratio higher than the averages found in previous studies (Wang et al., 2006;Whiteman et al., 2010). The increased higher risk observed in northeast Thailand may reflect the predominance of other risk factors in this population. Other variables, such as a family history of cancer in a first-degree relationship and lifestyle habits such as the diet of Thai people were associated with EC, and the magnitude of these effects was strong enough to achieve statistical significance (Boonyaphiphat et al., 2002). This study also investigated a possible correlation between the presence of the major virulence factors of H. pylori and EC. Our study analyzed the presence of the most prevalent virulence gene of H. pylori in 123 EC patients who were H. pylori-positive. The most prevalent of the virulence genes was CagA, which was found in 24% of the EC patients. CagA is in fact one of the most studied virulence genes of H. pylori. This protein has a molecular size of 138 kDa and is found in the CagA-PAI. H. pylori strains with CagA-PAI are more pathogenic than strains without CagA-PAI (Fischer, 2011). This study showed that CagA-positive H. pylori strains were significantly associated with EC (p-value < .001). More importantly, the results showed that EC patients with CagA-positive H. pylori strains had a significantly higher odds ratio and a statistically significant risk of developing ESCC (OR adj , 1.9; 95% CI, 1.76-4.77). However, we found no significant association between H. pylori or CagA positivity and EAC. On the contrary, H. pylori-CagA positivity tended to decrease the risk of EAC. A previous population-based study found that CagA positivity was associated with an increased risk of ESCC (OR adj , 2.1; 95% CI, 1.1-4.0) (Ye et al., 2004). A prospective study in China reported similar results (Wang et al., 2006). The results also agree with previous reports of CagA positivity in more than 60% of cases in East Asian countries, including Thailand (Chomvarin et al., 2008). Several studies have indicated that CagA-positive H. pylori strains are more virulent than CagA-negative strains. Moreover, the differences between the CagA genotypes are associated with the severity of human cancer. The CagA toxin plays a very important role in carcinogenesis in the stomach and other organs when CagA-synthesising H. pylori is detected. A specific system of CagA genes encodes the synthesis of the CagA toxin, which demonstrates the properties of reorganizing the cytoskeleton and the cell shape. Moreover, the toxin controls the transcription and proliferation of the cell, which leads to a chronic inflammatory reaction (Hatakeyama, 2017;Wroblewski et al., 2016). Many recent studies of animal and cell culture models indicated the likely role of CagA and Cag-PAI, such as EPIYA, in human gastric cancer associated with H. pylori infection (Sukri et al., 2020). This study is one of the very few studies that documented H. pylori testing of esophageal tissue specimens. H. pylori is a common found in the superficial mucosal layer of the stomach and also in the esophagus. There is some evidence that H. pylori plays a role in the progression of GERD to Barrett's esophagus to dysplasia to EC (Kountouras et al., 2012). The evidence specifically found that H. pylori may be involved in the pathophysiology of GERD via diverse mechanisms such as induction of mediators, cytokines and nitric oxide, which may disturb the lower esophageal sphincter and directly injure the esophageal mucosa due to bacterial products (Kountouras et al., 2006;Zhao et al., 2020). Another study suggested that H. pylori infection activates NF-κB, an oxidant-sensitive transcription regulator of the inducible expression of inflammatory genes, including COX-2, which regulates gastrointestinal neoplasm cell growth and proliferation. Specifically, H. pylori infection promotes the expression of NF-κB and COX-2 in esophageal epithelial cells, playing a role in the inflammatory process associated with BE and esophageal oncogenesis (Polyzos et al., 2018). Besides CagA, another possible cause of the activation of NF-κB and IL-8 secretion is esophageal metaplasia through chronic inflammation (Glocker et al., 1998;Li et al., 2014). A study inferred that H. pylori infection can cause ESCC through gastric atrophy, which may promote the excessive growth of bacteria and increase the production of endogenous nitrosamine, which may lead to ESCC (Iijima et al., 2010). The different virulence factors of H. pylori strains are regarded as important in causing various types of EC cancer in infected individuals. CagA is considered the main major virulence marker of H. pylori infection. Therefore, the combination of the virulence gene of H. pylori, the host factors and the environmental factors should be investigated simultaneously. In addition, these findings again emphasize the importance of CagA positivity in the pathogenesis of H. pylori infection associated with EC in the Thai population. Regarding other factors related to EC disease, the lifestyle may affect the severity of the disease, such as the use of medicinal plants in food and the diets and habits of the different populations. Further studies are needed to explore the mechanism by which CagA-positive H. pylori is inversely associated with the risk of EAC and to confirm the positive association between CagA-positive H. pylori and ESCC. Especially, the Cag-PAI rearrangement and the CagA polymorphism should be elucidated in different geographical regions for more molecular epidemiological data and pathogenesis. Moreover, both co-infection with H. pylori and Campylobacter spp and a single bacterial infection were also significantly associated with EC. This may indicate that these two bacteria are involved in the pathogenesis of EC. This study also had several limitations. A main limitation concerns the retrospective case-control design, from which a cause-effect relationship between H. pylori infection and EC risk was established. In this study, the recall bias of the measured risk factors might have affected our results, although efforts were made to minimize this bias. Moreover, we could not obtain enough information about the sociodemographic characteristics and the dietary and other lifestyle risk factors of the subjects. These factors could have influenced the EC outcome. Another limitation of the study was that the selected hospital controls might not have accurately reflected the exposure distributions of the source population and thus, might have affect the results.
In summary, the findings in this study give more evidence of the association of H. pylori infection and CagA-positive with other well-known risk factors such as tobacco smoking, alcohol drinking and personal history of GERD to elucidate the effect of those risk factors in the prevalence of H. pylori in EC. Our study showed a significant effect of H. pylori infection as risk of EC. Likewise, smoking status, alcohol use and GERD were associated with EC in our sample, the magnitude of these effects were strong enough to achieve statistical significance. Therefore, our results may have policy implications in the realize that population education and awareness of the results. In addition, the results in this study might have useful roles of the clinical helps to effectively diagnose and treat EC patients by understanding the trend of H. pylori infection in Thailand.

Author Contribution Statement
Poosari A , Nutravong T , Namwat W, Sa-ngiamwibool P contributed to the conception and designed the experiments. Ungareewittaya P, Poosari A, Sangiamwibool P pathological data and tissue esophageal cancer (FFPE) sample. Poosari A , Nutravong T performed the experiments and reviewed statistic analyzed the data. Poosari A , Nutravong T, Boonyanugomol W wrote the initial draft of the manuscript. All of the authors contributed to the editing and approved the final version of this manuscript..