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Table S1. Differentially methylated CpGs of non-clustered PCDHs by H. pylori eradication in the whole-genome methylation profiling. Table S2. The association between major characteristics and methylation of four PCDH genes. Table S3. Associations between DNA methylation levels and expression. Table S4. PCR primers and experiment condition of candidate genes. Details for immunohistochemical staining

Figure S1. Representative pictures of immunohistochemical staining for PCDH10 and PCDH17 (20X) PCDH10 and PCDH17 were expressed in the membrane and cytoplasm of epithelial and some stromal cell. (a, b) Representative pictures of immunohistochemical staining for PCDH10 negative and positive expression. (c, d) Representative pictures of immunohistochemical staining for PCDH17 negative and positive expression.

The association of evolutions of Ind DYS/DYS subjects with COX-2 expression changes.

To explore the epigenetic mechanisms underlying the effects of anti-Helicobacter pylori (H. pylori) alone and combined with COX-2 inhibitor (celecoxib), we dynamically evaluated the associations between COX-2 methylation alterations and gastric lesion evolution during the process of interventions. In a total of 809 trial participants COX-2 methylation levels were quantitatively detected before and after treatment. The self-comparison at the same stomach site for each subject showed significant methylation alteration differences among intervention groups (P < 0.001). With placebo group as reference, COX-2 methylation levels were decreased in anti-H. pylori [OR, 3.30; 95% confidence interval (CI), 2.16–5.02], celecoxib (OR, 2.04; 95% CI, 1.36–3.07), and anti-H. pylori followed by celecoxib (OR, 2.10; 95% CI, 1.38–3.17) groups. When stratified by baseline histology, the three active arms significantly decreased COX-2 methylation levels in indefinite dysplasia/dysplasia subjects, and ORs were 3.65 (95% CI, 1.96–6.80) for anti-H. pylori, 2.43 (95% CI 1.34–4.39) for celecoxib, and 2.80 (95% CI, 1.52–5.15) for anti-H. pylori followed by celecoxib, respectively. No additive effect on COX-2 methylation was found for anti-H. pylori followed by celecoxib than two treatments alone. Compared with subjects without methylation reduction, higher opportunity for gastric lesion regression was found in subjects with decreased COX-2 methylation levels, especially for indefinite dysplasia/dysplasia subjects (OR, 1.92; 95% CI, 1.03–3.60). These findings suggest that anti-H. pylori or celecoxib treatment alone could decrease COX-2 methylation levels in gastric mucosa. COX-2 methylation alteration was associated with the regression of indefinite dysplasia/dysplasia, which might serve as a potential biomarker for chemoprevention efficacy. Cancer Prev Res; 9(6); 484–90. ©2016 AACR.

Supplementary Table 2 from Manganese Superoxide Dismutase Polymorphism and Risk of Gastric Lesions, and Its Effects on Chemoprevention in a Chinese Population

Supplementary Table 1 from Manganese Superoxide Dismutase Polymorphism and Risk of Gastric Lesions, and Its Effects on Chemoprevention in a Chinese Population

Background: Genetic polymorphisms of Toll-like receptors (TLR) may influence the outcome of Helicobacter pylori infection and play important roles in gastric carcinogenesis. To screen the genetic variants of TLR2 and TLR5, and evaluate their associations with gastric cancer (GC) and its precursors, a population-based study was conducted in Linqu County, Shandong Province, China.Methods: Genetic variants were identified by PCR-based denaturing high-performance liquid chromatography and PCR-restriction fragment length polymorphism analysis in 248 GC cases, 846 subjects with advanced gastric lesions including 350 dysplasia and 496 intestinal metaplasia, and 496 superficial gastritis/mild chronic atrophic gastritis controls.Results: Nine allelic variants each were detected within the promoter and exons of TLR2 and TLR5. Among those, TLR2 c. −196 to −174 del carriers (ins/del+del/del) showed a significantly decreased risk of GC (adjusted OR, 0.66; 95% CI: 0.48–0.90), whereas TLR5 rs5744174 C carriers (TC+CC) had an increased risk of GC (OR, 1.43; 95% CI: 1.03–1.97). Further analysis indicated an elevated risk of GC in subjects with the TLR5 rs5744174 TC+CC genotype and H. pylori infection (OR, 3.35; 95% CI: 2.13–5.26), and a significant interaction between rs5744174 and H. pylori infection was observed (OR, 2.15; 95% CI: 1.12–4.16).Conclusion: These findings suggest that TLR2 c. −196 to −174 ins > del, TLR5 rs5744174 and interaction between rs5744174 and H. pylori infection were associated with the development of GC.Impact:TLR2 and TLR5 polymorphisms may play important roles in the process of H. pylori-related gastric carcinogenesis. Cancer Epidemiol Biomarkers Prev; 20(12); 2594–602. ©2011 AACR.

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Background: Manganese superoxide dismutase is the primary antioxidant enzyme in the mitochondria and is involved in carcinogenesis. To investigate the association between MnSOD Val16Ala polymorphism and risk of advanced gastric lesions, and its effects on chemoprevention, a population-based study was conducted in Linqu, a high-risk area of gastric cancer in China.Methods: Genotypes were determined by PCR-RFLP analysis in 3,355 subjects with the baseline histopathologic diagnosis in 1994, and 2,758 of these subjects received subsequent three interventions including vitamin supplementation for 7.3 years. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by unconditional logistic regression model.Results: We found an increased risk of dysplasia in subjects with the Val/Ala+Ala/Ala genotype (OR, 1.31; 95% CI, 1.02-1.68) compared with the Val/Val genotype. Stratified analysis indicated that a significantly elevated risk of intestinal metaplasia (OR, 3.40; 95% CI, 2.64-4.38) or dysplasia (OR, 4.01; 95% CI, 2.79-5.74) was found in subjects carrying the Val/Ala+Ala/Ala genotype and Helicobacter pylori infection, and an interaction between this genotype and a high serum H. pylori IgG titer (>2.94) on the risk of dysplasia was observed (Pinteraction = 0.01). Furthermore, an elevated chance for regression of gastric lesions was observed in subjects with the Val/Ala+Ala/Ala genotype and high IgG titer in an intervention trial with vitamin supplementation (OR, 2.45; 95% CI, 1.37-4.38).Conclusions: These findings suggest that Val16Ala polymorphism may play an important role in development of advanced gastric lesions and modify the effect of vitamin supplementation on the evolution of gastric lesions.Impact: Val16Ala polymorphism is related to gastric cancer development. Cancer Epidemiol Biomarkers Prev; 19(4); 1089–97. ©2010 AACR.

Supplementary Table 3 from Manganese Superoxide Dismutase Polymorphism and Risk of Gastric Lesions, and Its Effects on Chemoprevention in a Chinese Population

Background and Aim: Methylation alterations may be involved in Helicobacter pylori-associated gastric carcinogenesis. This study aims to explore the potential H.pylori-associated methylation biomarkers in blood leukocyte and gastric mucosa. Methods: Five candidate H.pylori-associated aberrant methylation genes were selected from the previous genome-wide profiling panels and validated in blood leukocyte and gastric mucosa in multi-stages (case-control validation between H.pylori positive and negative subjects and self-control validation before and after anti-H.pylori treatment). Results: GNAS methylation level was decreased in blood leukocyte (62.07% v.s. 46.33%, p

Background and aim Dietary strategies that contribute to reducing incidence of Helicobacter pylori infection without negative side effects are highly desirable owing to worldwide bacterial prevalence and carcinogenesis potential. The aim of this study was to determine dosage effect of daily cranberry consumption on H. pylori suppression over time in infected adults to assess the potential of this complementary management strategy in a region with high gastric cancer risk and high prevalence of H. pylori infection. Methods This double‐blind, randomized, placebo‐controlled trial on 522 H. pylori‐positive adults evaluated dose–response effects of proanthocyanidin‐standardized cranberry juice, cranberry powder, or their placebos on suppression of H. pylori at 2 and 8 weeks by 13C‐urea breath testing and eradication at 45 days post‐intervention. Results H. pylori‐negative rates in placebo, low‐proanthocyanidin, medium‐proanthocyanidin, and high‐proanthocyanidin cranberry juice groups at week 2 were 13.24%, 7.58%, 1.49%, and 13.85% and at week 8 were 7.35%, 7.58%, 4.48%, and 20.00%, respectively. Consumption of high‐proanthocyanidin juice twice daily (44 mg proanthocyanidin/240‐mL serving) for 8 weeks resulted in decreased H. pylori infection rate by 20% as compared with other dosages and placebo (P

Objective: Upper gastrointestinal (UGI) cancers, predominantly gastric cancer (GC) and esophageal cancer (EC), are malignant tumor types with high morbidity and mortality rates. Accumulating studies have focused on metabolomic profiling of UGI cancers in recent years. In this systematic review, we have provided a collective summary of previous findings on metabolites and metabolomic profiling associated with GC and EC. Methods: A systematic search of three databases (Embase, PubMed, and Web of Science) for molecular epidemiologic studies on the metabolomic profiles of GC and EC was conducted. The Newcastle–Ottawa Scale (NOS) was used to assess the quality of the included articles. Results: A total of 52 original studies were included for review. A number of metabolites were differentially distributed between GC and EC cases and non-cases, including those involved in glycolysis, anaerobic respiration, tricarboxylic acid cycle, and protein and lipid metabolism. Lactic acid, glucose, citrate, and fumaric acid were among the most frequently reported metabolites of cellular respiration while glutamine, glutamate, and valine were among the most commonly reported amino acids. The lipid metabolites identified previously included saturated and unsaturated free fatty acids, aldehydes, and ketones. However, the key findings across studies to date have been inconsistent, potentially due to limited sample sizes and the majority being hospital-based case-control analyses lacking an independent replication group. Conclusions: Studies on metabolomics have thus far provided insights into etiological factors and biomarkers for UGI cancers, supporting the potential of applying metabolomic profiling in cancer prevention and management efforts.

Background The effectiveness of endoscopic screening on gastric cancer has not been widely investigated in China and the screening interval of repeated screening has not been determined. Methods In a population-based prospective study, we included 375,800 individuals, 14,670 of whom underwent endoscopic screening (2012–2018). We assessed the associations between endoscopic screening and risk of incident gastric cancer and gastric cancer-specific mortality, and examined changes in overall survival and disease-specific survival following screening. The optimal screening interval for repeated endoscopy for early detection of gastric cancer was explored. Results Ever receiving endoscopic screening significantly decreased the risk of invasive gastric cancer (age- and sex-adjusted relative risk [RR] 0.69, 95 % confidence interval [CI] 0.52–0.92) and gastric cancer-specific mortality (RR 0.33, 95 %CI 0.20–0.56), particularly for noncardia gastric cancer. Repeated screening strengthened the beneficial effect on invasive gastric cancer-specific mortality of one-time screening. Among invasive gastric cancers, screening-detected individuals had significantly better overall survival (RR 0.18, 95 %CI 0.13–0.25) and disease-specific survival (RR 0.18, 95 %CI 0.13–0.25) than unscreened individuals, particularly for those receiving repeated endoscopy. For individuals with intestinal metaplasia or low grade intraepithelial neoplasia, repeated endoscopy at an interval of Conclusion Endoscopic screening prevented gastric cancer occurrence and death, and improved its prognosis in a population-based study. Repeated endoscopy enhanced the effectiveness. Screening interval should be based on gastric lesion severity.

Background Molecular features underlining the multistage progression of gastric lesions and development of early gastric cancer (GC) are poorly understood, restricting the ability to GC prevention and management. Methods We portrayed proteomic landscape and explored proteomic signatures associated with progression of gastric lesions and risk of early GC. Tissue proteomic profiling was conducted for a total of 324 subjects. A case-control study was performed in the discovery stage (n=169) based on populations from Linqu, a known high-risk area for GC in China. We then conducted two-stage validation, including a cohort study from Linqu (n = 56), with prospective follow-up for progression of gastric lesions (280-473 days), and an independent case-control study from Beijing (n = 99). Findings There was a clear distinction in proteomic features for precancerous gastric lesions and GC. We derived four molecular subtypes of gastric lesions and identified subtype-S4 with the highest progression risk. We found 104 positively-associated and 113 inversely-associated proteins for early GC, with APOA1BP, PGC, HPX and DDT associated with the risk of gastric lesion progression. Integrating these proteomic signatures, the ability to predict progression of gastric lesions was significantly strengthened (areas-under-the-curve=0.88 (95%CI: 0.78-0.99) vs. 0.56 (0.36-0.76), Delong's P = 0.002). Immunohistochemistry assays and examination at mRNA level validated the findings for four proteins. Interpretation We defined proteomic signatures for progression of gastric lesions and risk of early GC, which may have translational significance for identifying particularly high-risk population and detecting GC at an early stage, improving potential for targeted GC prevention. Funding The funders are listed in the Acknowledgement.

ObjectiveGastrointestinal microbiota may be involved in Helicobacter pylori-associated gastric cancer development. The aim of this study was to explore the possible microbial mechanisms in gastric carcinogenesis and potential dysbiosis arising from H. pylori infection.DesignDeep sequencing of the microbial 16S ribosomal RNA gene was used to investigate alterations in paired gastric biopsies and stool samples in 58 subjects with successful and 57 subjects with failed anti-H. pylori treatment, relative to 49 H. pylori negative subjects.ResultsIn H. pylori positive subjects, richness and Shannon indexes increased significantly (both pH. pylori positive mucosa was associated with advanced gastric lesions (chronic atrophic gastritis and intestinal metaplasia/dysplasia) and could be reversed by eradication. Strong coexcluding interactions between Helicobacter and Fusobacterium, Neisseria, Prevotella, Veillonella, Rothia were found only in advanced gastric lesion patients, and were absent in normal/superficial gastritis group. Changes in faecal microbiota included increased Bifidobacterium after successful H. pylori eradication and more upregulated drug-resistant functional orthologs after failed treatment.ConclusionH. pylori infection contributes significantly to gastric microbial dysbiosis that may be involved in carcinogenesis. Successful H. pylori eradication potentially restores gastric microbiota to a similar status as found in uninfected individuals, and shows beneficial effects on gut microbiota.

Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer death worldwide (1). It is imperative to identify patients with GC at a high risk for poor prognosis, so that timely and appropriate treatment strategy can be applied. Although a considerable proportion of patients are diagnosed at an advanced stage, patient outcomes can vary significantly even for patients with similar clinical features. The host heterogeneity in somatic or germline changes may have led to different prognosis. Several studies have examined both genetic and epigenetic alterations as potential prognostic factors for GC. Alternative patterns of messenger RNA (mRNA) expression involved in cycle regulation (2), cell adhesion (3), angiogenesis (4), and tumor carcinogenesis (5) have been reported to play an important role in forecasting survival outcome of GC. One study examined the gene expression profile of 65 patients with GC based on DNA microarray data and reported 6 mRNAs associated with GC prognosis (6). Another study reported 3 mRNAs associated with GC survival based on DNA microarray data of 21 patients with GC (7). A recent study reported 25 mRNAs associated with GC prognosis using DNA microarray data of 78 patients with GC (8). These studies were limited by either small sample size or lacking suitable validation datasets, restricting the possible application of the reported mRNAs associated with GC prognosis in clinical practices. Therefore, it is imperative to identify novel mRNA signatures robustly associated with GC survival. The booming development in high-throughput transcriptome sequencing and microarray technologies have provided opportunities to establish mRNA signatures associated with GC survival. In the present study, we took advantage of the publicly available databases to establish novel mRNA signatures, which may predict GC prognosis. Discovery of potential mRNAs was conducted among 441 patients with GC based on the Cancer Genome Atlas (TCGA), with information on clinical characteristics and vital status. Gene ontology functional enrichment analysis and pathway enrichment analysis were conducted to interrogate the possible biological functions. The independent external microarray data from the Gene Expression Omnibus (GEO) was used to validate these candidate mRNAs. A risk score model was then constructed using the validated mRNAs to testify the potential predictive value of integrated mRNA signature for GC prognosis.

Gastric cancer (GC) is one of the major cancers in China and all over the world. Most GCs are diagnosed at an advanced stage with unfavorable prognosis. Along with some other countries, China has developed the government-funded national screening programs for GC and other major cancers. GC screening has been shown to effectively decrease the incidence of and mortality from GC in countries adopting nationwide screening programs (Japan and Korea) and in studies based on selected Chinese populations. The screening of GC relies mostly on gastroendoscopy, the accuracy, reliability and safety of which have been indicated by previous studies. However, considering its invasive screening approach, requirements on skilled endoscopists and pathologists, and a high cost, developing noninvasive methods to amend endoscopic screening would be highly needed. Numerous studies have examined biomarkers for GC screening and the combination of biomarkers involving pepsinogen, gastrin, and Helicobacter pylori antibodies has been proposed for risk stratification, seeking to narrow down the high-risk populations for further endoscopy. Despite all the achievements of endoscopic screening, evidence on appropriate screening age, intervals for repeated screening, novel biomarkers promoting precision prevention, and health economics need to be accumulated to inform policymakers on endoscopic screening in China. With the guide of Health China 2030 Planning Outline, we have golden opportunities to promote prevention and control of GC. In this review, we summarize the characteristics of screening programs in China and other East Asian countries and introduce the past and current approaches and strategies for GC screening, aiming for featuring the latest advances and key challenges, and illustrating future visions of GC screening.

Background Few studies have examined prognostic outcomes-associated molecular signatures other than overall survival (OS) for gastric cancer (GC). We aimed to identify DNA methylation biomarkers associated with multiple prognostic outcomes of GC in an epigenome-wide association study. Methods Based on the Cancer Genome Atlas (TCGA), DNA methylation loci associated with OS (n = 381), disease-specific survival (DSS, n = 372), and progression-free interval (PFI, n = 383) were discovered in training set subjects (false discovery rates P-values n = 247). Gene set enrichment analyses were conducted to explore the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways simultaneously enriched for multiple GC prognostic outcomes. Methylation correlated blocks (MCBs) were identified for co-methylation patterns associated with GC prognosis. Based on key CpGs, risk score models were established to predict four prognostic outcomes. Spearman correlation analyses were performed between key CpG sites and their host gene mRNA expression. Results We newly identified DNA methylation of seven CpGs significantly associated with OS, DSS, and PFI of GC, including cg10399824 (GRK5), cg05275153 (RGS12), cg24406668 (MMP9), cg14719951(DSC3), and cg25117092 (MED12L), and two in intergenic regions (cg11348188 and cg11671115). Except cg10399824 and cg24406668, five of them were also significantly associated with DFI of GC. Neuroactive ligand-receptor interaction pathway was suggested to play a key role in the effect of DNA methylation on GC prognosis. Consistent with individual CpG-level association, three MCBs involving cg11671115, cg14719951, and cg24406668 were significantly associated with multiple prognostic outcomes of GC. Integrating key CpG loci, two risk score models performed well in predicting GC prognosis. Gene body DNA methylation of cg14719951, cg10399824, and cg25117092 was associated with their host gene expression, whereas no significant associations between their host gene expression and four clinical prognostic outcomes of GC were observed. Conclusions We newly identified seven CpGs associated with OS, DSS, and PFI of GC, with five of them also associated with DFI, which might inform patient stratification in clinical practices.

In addition to Helicobacter pylori (H.pylori), gastric microbiota may be involved in carcinogenesis process. However, the longitudinal study to assess changes in the gastric microbiota associated with the development of gastric carcinogenesis is still limited. The aim of this study is to explore dynamic microbial alterations in gastric cancer (GC) development based on a 4-year endoscopic follow-up cohort in Linqu County, China. Microbial alterations were investigated by deep sequencing of the microbial 16S ribosomal RNA gene in 179 subjects with various gastric lesions, and validated in paired gastric biopsies prospectively collected before and after lesion progression and in non-progression controls. Significant differences were found in microbial diversity and community structure across various gastric lesions, with 62 candidate differential taxa between at least two lesion groups. Further validations identified Helicobacter, Bacillus, Capnocytophaga and Prevotella to be associated with lesion progression-to-dysplasia (DYS)/GC (all P < 0.05), especially for subjects progressing from intestinal metaplasia (IM) to DYS/GC. The combination of the four genera in a microbial dysbiosis index showed a significant difference after lesion progression-to-DYS/GC compared to controls (P = 0.027). The panel including the four genera identified subjects after progression-to-DYS/GC with an area under the receiver-operating curve (AUC) of 0.941. Predictive significance was found before lesion progression-to-DYS/GC with an AUC = 0.776 and an even better AUC (0.927) for subjects progressing from IM to DYS/GC. Microbiota may play different roles at different stages in gastric carcinogenesis. A panel of bacterial genera associated with gastric lesions may help to assess gastric microbial dysbiosis and show potential predictive values for lesion progression. Our findings provide new clues for the microbial mechanism of H.pylori-associated carcinogenesis.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase that has been described to be frequently mutated in gastrointestinal cancers. RNF43 downregulation was associated with distant metastasis, TNM stage and poorer survival in patients with gastric and colorectal cancers. Functional analysis has shown that overexpressed RNF43 negatively regulates Wnt signalling by ubiquitinating Frizzled receptors and targeting them for degradation and by sequestering T-cell factor 4 (TCF4) to the nuclear membrane, thereby inhibiting Wnt-mediated transcription. In the stomach, RNF43 overexpression was shown to impair stem-like properties and to be negatively correlated with expression of Wnt-target genes. In this study, we show that RNF43 knockdown enhances the tumourigenic potential of gastric and colorectal cancer cell lines in vitro and in vivo. Thus, loss of RNF43 leads to increased proliferation and anchorage-independent growth as well as increased invasive capacity. In a xenograft model, RNF43 depletion enhanced tumour growth. Furthermore, we established two mouse models in which mutations in the RING domain of RNF43 were introduced. In the intestine and colon, loss of Rnf43 did not induce changes in epithelial architecture or proliferation. In contrast, in the stomach, thickening of the mucosa, hyperplasia and cellular atypia were observed in these mice. Notably, this was independent of elevated Wnt signalling. Together, our results show that RNF43 plays a tumour suppressive role in gastric and colorectal cancer cells and that the loss of its function alters gastric tissue homeostasis in vivo.

Objective To identify serum biomarkers that may predict the short or long term outcomes of anti-Helicobacter pylori (H. pylori) treatment, a follow-up study was performed based on an intervention trial in Linqu County, China. Methods A total of 529 subjects were selected randomly from 1,803 participants to evaluate total anti-H. pylori immunoglobulin G (IgG) and 10 specific antibody levels before and after treatment at 1-, 2- and 7.3-year. The outcomes of anti-H. pylori treatment were also parallelly assessed by13C-urea breath test at 45-d after treatment and 7.3-year at the end of follow-up. Results We found the medians of anti-H. pylori IgG titers were consistently below cut-off value through 7.3 years in eradicated group, however, the medians declined in recurrence group to 1.2 at 1-year after treatment and slightly increased to 2.0 at 7.3-year. While the medians were significantly higher (>3.0 at 2- and 7.3-year) among subjects who failed the eradication or received placebo. For specific antibody responses, baseline seropositivities of FliD and HpaA were reversely associated with eradication failure [for FliD, odds ratio (OR)=0.44, 95% confidence interval (95% CI): 0.27-0.73; for HpaA, OR=0.32, 95% CI: 0.17-0.60]. The subjects with multiple positive specific antibodies at baseline were more likely to be successfully eradicated in a linear fashion (Ptrend=0.006). Conclusions Our study suggested that total anti-H. pylori IgG level may serve as a potential monitor of long-term impact on anti-H. pylori treatment, and priority forH. pylori treatment may be endowed to the subjects with multiple seropositive antibodies at baseline, especially for FliD and HapA.

Background: Telomeres have long been found to be involved in cancer development, while little was known about the dynamic changes of telomere length in carcinogenesis process.Methods: The present study longitudinally investigated telomere alterations of cell-free DNA (cfDNA) in 86 gastric cancer (GC) subjects recruited through a 16-year prospective cohort with 2–4 serums collected before each GC-diagnosis from baseline and three follow-up time-points (a total of 276 samples). As the control, 86 individual-matched cancer-free subjects were enrolled with 276 serums from the matched calendar year.Results: In the 73 pairs of baseline serums from GC and control subjects, shortened telomeres showed increased subsequent GC risk [odds ratio (OR) = 9.17, 95% CI: 2.72–31.25 for 1 unit shortening]. In each baseline gastric lesion category, higher risks of GC progression were also found with shortened cfDNA telomeres; ORs per 1 unit shortening were 6.99 (95% CI: 1.63–30.30) for mild gastric lesions, 6.06 (95% CI: 1.89–19.61) for intestinal metaplasia and 15.63 (95% CI: 1.91–125.00) for dysplasia. With all measurements from baseline and follow-up time-points, shortened telomeres also showed significant association with GC risk (OR = 7.37, 95% CI: 2.06–26.32 for 1 unit shortening). In temporal trend analysis, shortened telomeres were found in GC subjects compared to corresponding controls more than 3 years ahead of GC-diagnosis (most P < 0.05), while no significant difference was found between two groups within 3 years approaching to GC-diagnosis.Conclusion: Our findings suggest that telomere shortening may be associated with gastric carcinogenesis, which supports further etiological study and potential biomarker for risk stratification.

Objective To assess the effects of Helicobacter pylori treatment, vitamin supplementation, and garlic supplementation in the prevention of gastric cancer. Design Blinded randomized placebo controlled trial. Setting Linqu County, Shandong province, China. Participants 3365 residents of a high risk region for gastric cancer. 2258 participants seropositive for antibodies to H pylori were randomly assigned to H pylori treatment, vitamin supplementation, garlic supplementation, or their placebos in a 2×2×2 factorial design, and 1107 H pylori seronegative participants were randomly assigned to vitamin supplementation, garlic supplementation, or their placebos in a 2×2 factorial design. Interventions H pylori treatment with amoxicillin and omeprazole for two weeks; vitamin (C, E, and selenium) and garlic (extract and oil) supplementation for 7.3 years (1995-2003). Main outcome measures Primary outcomes were cumulative incidence of gastric cancer identified through scheduled gastroscopies and active clinical follow-up through 2017, and deaths due to gastric cancer ascertained from death certificates and hospital records. Secondary outcomes were associations with other cause specific deaths, including cancers or cardiovascular disease. Results 151 incident cases of gastric cancer and 94 deaths from gastric cancer were identified during 1995-2017. A protective effect of H pylori treatment on gastric cancer incidence persisted 22 years post-intervention (odds ratio 0.48, 95% confidence interval 0.32 to 0.71). Incidence decreased significantly with vitamin supplementation but not with garlic supplementation (0.64, 0.46 to 0.91 and 0.81, 0.57 to 1.13, respectively). All three interventions showed significant reductions in gastric cancer mortality: fully adjusted hazard ratio for H pylori treatment was 0.62 (95% confidence interval 0.39 to 0.99), for vitamin supplementation was 0.48 (0.31 to 0.75), and for garlic supplementation was 0.66 (0.43 to 1.00). Effects of H pylori treatment on both gastric cancer incidence and mortality and of vitamin supplementation on gastric cancer mortality appeared early, but the effects of vitamin supplementation on gastric cancer incidence and of garlic supplementation only appeared later. No statistically significant associations were found between interventions and other cancers or cardiovascular disease. Conclusions H pylori treatment for two weeks and vitamin or garlic supplementation for seven years were associated with a statistically significant reduced risk of death due to gastric cancer for more than 22 years. H pylori treatment and vitamin supplementation were also associated with a statistically significantly reduced incidence of gastric cancer. Trial registration ClinicalTrials.gov NCT00339768.

Importance Metabolic deregulation plays an important role in gastric cancer (GC) development. To date, no studies have comprehensively explored the metabolomic profiles along the cascade of gastric lesions toward GC. Objective To draw a metabolic landscape and define metabolomic signatures associated with the progression of gastric lesions and risk of early GC. Design, setting, and participants A 2-stage, population-based cohort study was initiated in 2017 in Linqu County, Shandong Province, China, a high-risk area for GC. Prospective follow-up was conducted during the validation stage (June 20, 2017, to May 27, 2020). A total of 400 individuals were included based on the National Upper Gastrointestinal Cancer Early Detection Program in China. The discovery stage involved 200 individuals with different gastric lesions or GC (high-grade intraepithelial neoplasia or invasive GC). The validation stage prospectively enrolled 152 individuals with gastric lesions who were followed up for 118 to 1063 days and 48 individuals with GC. Exposures Metabolomic profiles and metabolite signatures were examined based on untargeted plasma metabolomics assay. Main outcomes and measures The risk of GC overall and early GC (high-grade intraepithelial neoplasia), and progression of gastric lesions. Results Of the 400 participants, 124 of 200 (62.0%) in the discovery set were men; mean (SD) age was 56.8 (7.5) years. In the validation set, 136 of 200 (68.0%) were men; mean (SD) age was 57.5 (8.1) years. Distinct metabolomic profiles were noted for gastric lesions and GC. Six metabolites, including α-linolenic acid, linoleic acid, palmitic acid, arachidonic acid, sn-1 lysophosphatidylcholine (LysoPC)(18:3), and sn-2 LysoPC(20:3) were significantly inversely associated with risk of GC overall and early GC (high-grade intraepithelial neoplasia). Among these metabolites, the first 3 were significantly inversely associated with gastric lesion progression, especially for the progression of intestinal metaplasia (α-linolenic acid: OR, 0.42; 95% CI, 0.18-0.98; linoleic acid: OR, 0.43; 95% CI, 0.19-1.00; and palmitic acid: OR, 0.32; 95% CI, 0.13-0.78). Compared with models including only age, sex, Helicobacter pylori infection, and gastric histopathologic findings, integrating these metabolites significantly improved the performance for predicting the progression of gastric lesions (area under the curve [AUC], 0.86; 95% CI, 0.70-1.00 vs AUC, 0.69; 95% CI, 0.50-0.88; P = .02) and risk of early GC (AUC, 0.83; 95% CI, 0.58-1.00 vs AUC, 0.61; 95% CI, 0.31-0.91; P = .03). Conclusions and relevance This study defined metabolite signatures that might serve as meaningful biomarkers for assessing high-risk populations and early diagnosis of GC, possibly advancing targeted GC prevention and control.

// Yang Zhang 1, * , Xin-ran Zhang 1, * , Jong-lyul Park 2, * , Jong-hwan Kim 2 , Lian Zhang 1 , Jun-ling Ma 1 , Wei-dong Liu 3 , Da-jun Deng 4 , Wei-cheng You 1 , Yong-sung Kim 2 , Kai-feng Pan 1 1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Cancer Epidemiology, Peking University Cancer Hospital and Institute, Beijing, China 2 Medical Genomic Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea 3 Department of Medical Oncology, Healthy Bureau of Linqu County, Shandong, China 4 Department of Cancer Etiology, Peking University Cancer Hospital and Institute, Beijing, China * These authors contributed equally to this work Correspondence to: Kai-feng Pan, email: pankaifeng2002@yahoo.com Yong-sung Kim, email: yongsung@kribb.re.kr Keywords: H. pylori, gastric mucosa, blood leukocyte, methylation array Received: December 23, 2015 Accepted: April 24, 2016 Published: May 19, 2016 ABSTRACT Purpose: To investigate Helicobacter pylori (H.pylori) associated genome-wide aberrant methylation patterns in gastric mucosa and blood leukocyte DNA, a population-based study was conducted in Linqu County. Results: A total of 3000 and 386 CpGs were differentially methylated after successful H.pylori eradication in gastric mucosa and blood leukocyte DNA respectively, and 17 were the same alteration trend in the both tissues. The differentially methylated CpGs were located more frequently in promoters or CpG islands for gastric mucosa and gene body or open sea for blood leukocyte DNA. In eradicated gastric mucosa, the hypermethylated CpGs were enriched across inflammatory pathways, while the hypomethylated CpGs in tube morphogenesis, development and so on. The final validation found lower SPI1, PRIC285 and S1PR4 methylation levels in H.pylori positive subjects by case-control comparison, and increased methylation levels in H.pylori eradicated gastric mucosa by self-comparison. The Cancer Genome Atlas (TCGA) database analysis suggested that the up-regulation of the three genes by hypomethylation might be associated with gastric carcinogenesis. Experimental Design: Infinium HumanMethylation 450K BeadChip was used to compare methylation profiles prior to and after eradication treatment. The methylation levels of identified candidate differentially methylated genes before and after H.pylori eradication were further validated by two stages (Stage I: self-comparison of 16 subjects before and after anti- H.pylori treatment; Stage II: case-control comparison of 25 H.pylori positive and 25 negative subjects and self-comparison of 50 anti- H.pylori treated subjects). Conclusions: Novel H.pylori associated aberrant methylated genes were identified across the whole genome both in gastric mucosa and blood leukocyte DNA.

Background To explore the association between hypomethylation of repetitive elements ( LINE-1 , Sat2, and ALU ) in blood leukocyte DNA and risks of gastric lesions, and development of gastric cancer (GC), a population-based study was conducted in a high-risk area of GC in China. Materials Methylation levels were determined by MethyLight in 902 subjects with various gastric lesions from two cohort studies at baseline and 276 subjects with long-term follow-up data. Results The frequency of LINE-1 or Sat2 hypomethylation was significantly increased in subjects with dysplasia (DYS) compared with superficial gastritis/chronic atrophic gastritis. The odds ratios (ORs) were 2.22 [95% confidence interval (CI): 1.45–3.40] for LINE-1 and 1.58 (95% CI: 1.14–2.21) for Sat2 . A dose–response pattern was found for the risk of DYS and LINE-1 hypomethylation ( P -trend LINE-1 or Sat2 hypomethylation was higher in subjects with Helicobacter pylori infection. The ORs were 1.83 (95% CI: 1.12–2.99) for LINE-1 and 1.44 (95% CI: 1.01–2.05) for Sat2 . The follow-up data indicated that the risk of progression to GC was increased in intestinal metaplasia (IM) subjects with LINE-1 hypomethylation (OR=2.82; 95% CI: 1.17–6.77) or Sat2 hypomethylation (OR=2.78; 95% CI: 1.15–6.74). The risk of progression to GC was also increased in DYS subjects with Sat2 hypomethylation (OR=5.24; 95% CI: 2.00–13.74). Conclusions These findings suggest that hypomethylation of repetitive elements in blood leukocytes is associated with the risks of advanced gastric lesions and development of GC.

Background: Helicobacter pylori is the leading cause of gastric cancer, yet the majority of infected individuals will not develop neoplasia. Previously, we developed and replicated serologic H. pylori biomarkers for gastric cancer risk among prospective cohorts in East Asia and now seek to validate the performance of these biomarkers in identifying individuals with premalignant lesions. Methods: This cross-sectional study included 1,402 individuals from Linqu County screened by upper endoscopy. H. pylori protein-specific antibody levels were assessed using multiplex serology. Multivariable-adjusted logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for prevalent intestinal metaplasia, indefinite dysplasia, or dysplasia, compared with superficial or mild atrophic gastritis. Results: Compared with individuals seronegative to Omp and HP0305, individuals seropositive to both were seven times more likely to have precancerous lesions (OR, 7.43; 95% CI, 5.59–9.88). A classification model for precancerous lesions that includes age, smoking, and seropositivity to H. pylori, Omp, and HP0305 resulted in an area under the curve (AUC) of 0.751 (95% CI, 0.725–0.777), which is significantly better than the same model, including the established gastric cancer risk factor CagA (AUC, 0.718; 95% CI, 0.691–0.746, Pdifference = 0.0002). Conclusions: The present study of prevalent precancerous gastric lesions provides support for two new serum biomarkers of gastric cancer risk, Omp and HP 0305. Impact: Our results support further research into the serological biomarkers Omp and HP0305 as possible improvements over the established virulence marker CagA for identifying individuals with precancerous lesions in East Asia.

Nonclustered protocadherins (PCDH) family is a group of cell–cell adhesion molecules. We have found differentially methylated genes in the nonclustered PCDHs family associated with Helicobacter pylori (H. pylori) infection in prior genome-wide methylation analysis. To further investigate the methylation and expression of nonclustered PCDHs encoding genes in H. pylori--related gastric carcinogenesis process, four candidate genes including PCDH7, PCDH10, PCDH17, and PCDH20 were selected, which were reported to be tumor suppressors for digestive cancers. A total of 747 participants with a spectrum of gastric lesions were enrolled from a high-risk population of gastric cancer. Promoter methylation levels of four genes were significantly higher in H. pylori–positive subjects than the negative group (all P < 0.001). Elevated methylation levels of PCDH10 and PCDH17 were observed with the increasing severity of gastric lesions (both Ptrend < 0.001). In the protein expression analysis, PCDH17 expression was inversely associated with gastric lesions; the OR [95% confidence interval (CI)] was 0.49 (0.26–0.95) for chronic atrophic gastritis (CAG), 0.31 (0.15–0.63) for intestinal metaplasia, and 0.38 (0.19–0.75) for indefinite dysplasia and dysplasia, compared with superficial gastritis. In addition, PCDH10 expression was significantly lower in CAG (OR, 0.40; 95% CI, 0.24–0.68). The inverse association between methylation and protein expression of PCDH10 and PCDH17 was further supported when we explored the methylation and mRNA expression in The Cancer Genome Atlas database (all P < 0.001). Our study found elevated promoter methylation and decreased expression of PCDH10 and PCDH17 in advanced gastric lesions, suggesting that elevated PCDH10 and PCDH17 methylation may be an early event in gastric carcinogenesis. Cancer Prev Res; 11(11); 717–26. ©2018 AACR.

Helicobacter pylori (H. pylori) infection and cytokine-mediated inflammatory responses play important roles in gastric cancer (GC) pathogenesis. To investigate an association between genetic polymorphisms in interleukin (IL)-1β, IL-4R, IL-8, IL-10, IL-16, IL-18RAP, IL-22, and IL-32 and risks of GC and its precursors, a population-based study was conducted in Linqu County. Genotypes were determined by Sequenom MassARRAY platform in 132 GC cases and 1198 subjects with gastric lesions. The H. pylori status was determined by (13)C-urea breath test ((13)C-UBT) or enzyme-linked immunosorbent assay (ELISA). Among 11 candidate single nucleotide polymorphisms (SNPs), subjects carrying IL-18RAP rs917997 AA genotype were associated with risk of GC [adjusted odds ratio (OR) = 1.83, 95 % confidence interval (CI) 1.14-2.92] or chronic atrophic gastritis (CAG; OR = 1.55, 95 % CI 1.07-2.24). The risk of GC was also increased in subjects carrying IL-32 rs2015620 A allele (AA + AT; OR = 1.92, 95 % CI 1.09-3.39). Moreover, elevated risks of CAG (OR = 2.64, 95 % CI 1.89-3.69), intestinal metaplasia (IM; OR = 5.58, 95 % CI 3.86-8.05), and dysplasia (DYS; OR = 1.64, 95 % CI 1.18-2.26) were observed in subjects with IL-22 rs1179251 CC genotype. Stratified analysis indicated that risks of GC and its precursors were elevated in subjects with IL-32 rs2015620 A allele (AA + AT) or IL-22 rs1179251 CC genotype and H. pylori infection, and significant interactions between these two SNPs and H. pylori infection were found. These findings suggested that IL-18RAP rs917997, IL-32 rs2015620, IL-22 rs1179251, and interactions between these polymorphisms and H. pylori infection were associated with risks of gastric lesions. Genetic polymorphisms of interleukins may play crucial roles in H. pylori-induced gastric carcinogenesis.

The performance of diagnostic tests in intervention trials of Helicobacter pylori (H.pylori) eradication is crucial, since even minor inaccuracies can have major impact. To determine the cut-off point for 13C-urea breath test (13C-UBT) and to assess if it can be further optimized by serologic testing, mathematic modeling, histopathology and serologic validation were applied. A finite mixture model (FMM) was developed in 21,857 subjects, and an independent validation by modified Giemsa staining was conducted in 300 selected subjects. H.pylori status was determined using recomLine H.pylori assay in 2,113 subjects with a borderline 13C-UBT results. The delta over baseline-value (DOB) of 3.8 was an optimal cut-off point by a FMM in modelling dataset, which was further validated as the most appropriate cut-off point by Giemsa staining (sensitivity = 94.53%, specificity = 92.93%). In the borderline population, 1,468 subjects were determined as H.pylori positive by recomLine (69.5%). A significant correlation between the number of positive H.pylori serum responses and DOB value was found (rs = 0.217, P 13C-UBT in community-based studies, and a second method to determine H.pylori status for subjects with borderline value of 13C-UBT was necessary and recommended.

Gastric cancer (GC) is a highly aggressive cancer and a major cause of cancer-related deaths worldwide. Approximately half of the world's GC cases and deaths occur in china. GC presents challenges in early diagnosis and effective therapy due to a lack of understanding of the underlying molecular biology. The primary goals of this review are to outline current GC research in china and describe future trends in this field. Areas covered: This review mainly focuses on a series of GC-related advances China has achieved. Considerable progress has been made in understanding the role of H. pylori in GC by a series of population-based studies in well-established high-risk areas; A few germline and somatic alterations have been identified by 'omics' studies; Studies on the mechanisms of malignant phenotypes have helped us to form an in-depth understanding of GC and advance drug discovery. Moreover, identification of potential biomarkers and targeted therapies have facilitated the diagnosis and treatment of GC. However, many challenges remain. Expert commentary: To combat GC, sufficient funding is important. More attention should be paid on early diagnosis and the discovery of novel efficient biomarkers and the development of biomarker-based or targeted therapeutics in GC.

Objective To assess the effects of Helicobacter pylori treatment, vitamin supplementation, and garlic supplementation in the prevention of gastric cancer. Design Blinded randomized placebo controlled trial. Setting Linqu County, Shandong province, China. Participants 3365 residents of a high risk region for gastric cancer. 2258 participants seropositive for antibodies to H pylori were randomly assigned to H pylori treatment, vitamin supplementation, garlic supplementation, or their placebos in a 2×2×2 factorial design, and 1107 H pylori seronegative participants were randomly assigned to vitamin supplementation, garlic supplementation, or their placebos in a 2×2 factorial design. Interventions H pylori treatment with amoxicillin and omeprazole for two weeks; vitamin (C, E, and selenium) and garlic (extract and oil) supplementation for 7.3 years (1995-2003). Main outcome measures Primary outcomes were cumulative incidence of gastric cancer identified through scheduled gastroscopies and active clinical follow-up through 2017, and deaths due to gastric cancer ascertained from death certificates and hospital records. Secondary outcomes were associations with other cause specific deaths, including cancers or cardiovascular disease. Results 151 incident cases of gastric cancer and 94 deaths from gastric cancer were identified during 1995-2017. A protective effect of H pylori treatment on gastric cancer incidence persisted 22 years post-intervention (odds ratio 0.48, 95% confidence interval 0.32 to 0.71). Incidence decreased significantly with vitamin supplementation but not with garlic supplementation (0.64, 0.46 to 0.91 and 0.81, 0.57 to 1.13, respectively). All three interventions showed significant reductions in gastric cancer mortality: fully adjusted hazard ratio for H pylori treatment was 0.62 (95% confidence interval 0.39 to 0.99), for vitamin supplementation was 0.48 (0.31 to 0.75), and for garlic supplementation was 0.66 (0.43 to 1.00). Effects of H pylori treatment on both gastric cancer incidence and mortality and of vitamin supplementation on gastric cancer mortality appeared early, but the effects of vitamin supplementation on gastric cancer incidence and of garlic supplementation only appeared later. No statistically significant associations were found between interventions and other cancers or cardiovascular disease. Conclusions H pylori treatment for two weeks and vitamin or garlic supplementation for seven years were associated with a statistically significant reduced risk of death due to gastric cancer for more than 22 years. H pylori treatment and vitamin supplementation were also associated with a statistically significantly reduced incidence of gastric cancer. Trial registration ClinicalTrials.gov NCT00339768 .

Gastric cancer (GC) is a consequence of multifactorial and multistep processes. Helicobacter pylori (H. pylori) infection plays a crucial role in gastric carcinogenesis. Long non-coding RNAs (lncRNAs) have shown great potential as powerful cancer biomarkers. To investigate the possible roles of lncRNAs and H. pylori infection in GC development, we measured expression levels of three lncRNAs (H19, LINC00152, uc001lsz) in serum from a total of 285 Chinese participants using reverse transcription-quantitative polymerase chain reaction. We found significant associations between high expression of both H19 and LINC00152 in serum and increased risk of GC; the adjusted OR for H19 was 2.17 (95% CI: 1.21-3.88), and for LINC00152 was 2.09 (95% CI: 1.18-3.70). Further analyses indicated an elevated risk of GC in subjects with both high H19 expression and H. pylori infection (OR: 13.75, 95% CI: 4.75-39.84). Significant joint effect between LINC00152 and H. pylori infection on risk of GC was also found (OR: 17.49, 95% CI: 4.78-63.92). Serum H19 and LINC00152 may serve as potential biomarkers for diagnosis of GC, particularly for those with H. pylori infection.