Aberrant DNA methylation and expression of EYA4 in gastric cardia intestinal metaplasia

Background: Intestinal metaplasia (IM) of the gastric cardia is an important premalignant lesion. However, there is limited information concerning its epidemiological and molecular features. Herein, we aimed to provide an overview of the epidemiological data for gastric cardiac IM and evaluate the role of EYA transcriptional coactivator and phosphatase 4 (EYA4) as an epigenetic biomarker for gastric cardiac IM.
Methods: The study was conducted in the context of the gastric cardiac precancerous lesion program in southern China, which included 718 non-cancer participants, who undertook endoscopic biopsy and pathological examination in three endoscopy centers, between November 2018 and November 2021. Pyrosequencing and immunohistochemistry were performed to examine the DNA methylation status and protein expression level of EYA4.
Results: Gastric cardiac IM presented in 14.1% (101/718) of participants and was more common among older (>50 years; 22.0% [95% CI: 17.8–26.8]) than younger participants (≤50 years; 6.7% [95% CI: 4.5–9.9]; P < 0.001). IM was more common in male participants (16.9% [95% CI: 13.2–21.3] vs. 11.3% [95% CI: 8.3–15.1]; P = 0.04). Pyrosequencing revealed that IM tissues exhibited significantly higher DNA methylation levels in EYA4 gene than normal tissues (P = 0.016). Further, the protein expression level of EYA4 was reduced in IM and absent in intraepithelial neoplasia tissues compared to normal tissues (P < 0.001).
Conclusions: Detection rates of gastric cardiac IM increase with age and are higher in men. Our findings highlight the important role of promoter hypermethylation and downregulation of EYA4 in gastric cardiac IM development.

Keywords: DNA methylation, EYA4, gastric cardia, intestinal metaplasia

How to cite this article:
Li C, Liu Z, Xu G, Wu S, Peng Y, Wu R, Zhao S, Liao X, Lin R. Aberrant DNA methylation and expression of EYA4 in gastric cardia intestinal metaplasia. Saudi J Gastroenterol 2022;28:456-65
How to cite this URL:
Li C, Liu Z, Xu G, Wu S, Peng Y, Wu R, Zhao S, Liao X, Lin R. Aberrant DNA methylation and expression of EYA4 in gastric cardia intestinal metaplasia. Saudi J Gastroenterol [serial online] 2022 [cited 2022 Nov 24];28:456-65. Available from: https://www.saudijgastro.com/text.asp?2022/28/6/456/360457

Authors Chenxi Li, Zhaohui Liu, Guohua Xu and Shibin Wu are contributed equally to this work. > > > Authors Xiaoqi Liao and Runhua Lin are co-corresponding authors.

Introduction

Intestinal metaplasia (IM) of the stomach is characterized by the replacement of normal gastric epithelium and gastric glands by intestinal epithelium and intestinal glands. Histologically confirmed gastric IM often confers an increased risk of progression towards gastric cancer.[1],[2],[3] Thus, gastric IMs are generally considered to be precursor lesions for the development of dysplasia, and ultimately gastric carcinoma.[4] However, little attention has been given to this premalignant lesion in the gastric cardia. Limited data are available on the prevalence of IM in gastric cardia, especially in cancer-free subjects. Yet, these data may be informative for further molecular investigations. Given the preneoplastic nature of the lesion, it is crucial to improve our understanding of the regulation of genes contributing to the intestinal metaplastic phenotype. Hence, identifying informative biomarkers for gastric cardiac IM could be useful to guide further research regarding gastric cardiac carcinogenesis, that may have preventive implications.

DNA methylation is a key regulator of gene expression that does not alter the DNA sequence. Although it is well known that aberrant DNA methylation is associated with multiple cancer types,[5],[6],[7] there is less clarity regarding its role in early neoplastic progression. Indeed, these changes to DNA methylation are critical to understanding whether altered DNA methylation may contribute to premalignant lesion formation. Recent epigenetic studies have shown that altered DNA methylation occurs even in precancerous tissues, and these changes, therefore, may serve as promising early indicators of existing disease, and of risk prediction.[1] More recently, our methylation array-based (Illumina EPIC/850K array) study showed that gastric cardiac IM exhibits distinct DNA methylation profiling and identified some candidate genes that were significantly hypermethylated in their promoter regions in IM tissues. In this work, using a stringent filtering strategy, we found that the EYA4 promoter region exhibited the largest number of differentially methylated probes (DMPs) in gastric cardiac IM tissue. Thus, we hypothesized that gastric cardia IM presents as a common event in cancer-free subjects, and that promoter hypermethylation of EYA4 may reduce its expression level in gastric cardiac IM tissues.

For the present study, we aimed to: 1) investigate the detection rate of gastric cardiac IM in cancer-free individuals, 2) verify the DNA methylation level of candidate DMPs in the EYA4 promoter region, and 3) evaluate the protein expression level of EYA4 in gastric cardiac IM and normal gastric cardiac mucosa.

Patients and Methods

Biopsy specimens

Gastric cardiac biopsies were obtained from 718 participants undergoing upper endoscopy for gastrointestinal symptoms, between November 2018 and November 2021, in three centers (Shantou, Huizhou, and Shenzhen) in Guangdong Province, China. All the subjects included in this study were cancer-free at the time of endoscopic examination. Informed written consent was obtained from the participants according to the protocol approved by the Institutional Review Board of Shantou University Medical College (Approval Number: SUMC-2020-23); (Approval date: April 3, 2020). Upper endoscopic examinations were conducted by four experienced endoscopists (attending physician or above, having experiences of gastrointestinal endoscopy for at least 5 years). Biopsies of the endoscopically normal gastric cardiac mucosae were included in this study. Following histopathological assessment, we divided these biopsy samples into four histological categories, including normal gastric cardiac tissue, chronic carditis, chronic atrophic carditis, and IM tissue [Figure 1]a and [Figure 1]b. For bisulfite pyrosequencing, the following subjects were included: n = 10 (normal tissue, n = 5; IM tissue, n = 5). For the immunostaining of EYA4, the following samples were included: n = 88 (normal tissue, n = 38; IM tissue, n = 47; and IM with intraepithelial neoplasia [IEN], n = 3). A total of four IEN samples were used for histopathological analyses in our study, but finally only three IEN samples were available for immunohistochemistry (IHC).

Figure 1: Overview of gastric cardiac biopsy specimens and detection rate of gastric cardiac IM. (a) schematic diagram showing the collection of gastric cardiac tissues from cancer-free individuals. A total of 718 participants underwent upper endoscopy were recruited from three endoscopy centers (Shantou, Huizhou, and Shenzhen) in Guangdong Province (b) hematoxylin and eosin staining of representative gastric cardiac tissues from participants with different pathological changes. Scale bars, 50 μm (c) representative images of Alcian blue-periodic acid-Schiff (AB-PAS) staining and immunostaining of mucin-2 (MUC2) in gastric cardiac IM tissues. The AB staining method stains goblet cell mucus as blue color (as indicated by a closed red arrowhead show AB-positive cells); the open red arrowhead denotes epithelial cells expressing MUC2. Scale bars, 50 μm (d) pie chart depicting the detection rates of 718 individuals in our cohort depending on their pathological changes. Left panel: Histologically, gastric cardiac IM was found in 14.1% (101/718) cancer-free individuals. Right panel: A total of four subjects show low-grade intraepithelial neoplasia of the gastric cardiac mucosa in the context of IM. There is mild irregularity of the glands with crowding of epithelial cells. Most of the glandular cells show mucus depletion, the nuclei are elongated, hyperchromatic, and irregular in size. Scale bar, 50 μm (e) boxplot showing the median age in different histological categories. Statistical significance was analyzed by the Kruskal–Wallis test, followed by Dunnett's post hoc test for multiple comparisons between groups. The black horizontal dashed line indicates the median age of all the study participants. The upper and lower limits of the box represent 25th and 75th quartile age distribution with whiskers extending to 1.5 times the range from top/bottom of the box, the center line within the box corresponds to the median age in each group, outliers are not shown (f) line graph showing detection rates of gastric cardiac IM measured in different age and sex groups. Indicated P values were calculated by Pearson's chi-square test. Error bar represents the 95% confidence interval around the detection rate. The overall detection rate of gastric cardiac IM (14.1%) in study participants is depicted by the horizontal dashed black line. Source data (d and f) are provided in [Supplementary Table S2]

Click here to view

Histopathological evaluation

Briefly, the endoscopic specimens were fixed with 10% neutral formalin for 24 h, and then specimens were embedded in paraffin. Paraffin-embedded tissues were cut into 4-μm thick sections and stained with hematoxylin and eosin (HE). The pathological diagnosis was independently determined by two pathologists. For the specimens showing IM lesions with routine HE staining on initial examination, two additional tissue sections were stained with Alcian blue/periodic acid-Schiff (AB-PAS; Cat# BA-4121, Lot: C210401, BaSO Biotechnologies, China) staining, and immunohistochemical staining of mucin 2 (MUC2; goblet cell marker) for further validation [Figure 1]c.

In this study, we classified all the endoscopic specimens into four categories (normal gastric cardiac tissue, chronic carditis, atrophic carditis, and IM tissue) based on the following histological criteria.[8] Briefly, (a) normal gastric cardiac tissues, represented by normal morphology of the mucosa, namely, foveola, glands, gland necks, and stroma are well-preserved and keep their position and proportions intact. Inflammatory infiltration is minimal or absent. (b) Chronic carditis, characterized by the infiltration of the lamina propria by plasma cells and lymphocytes (with occasional formation of follicles) without glandular atrophy. (c) Chronic atrophic carditis, defined as the reduction or disappearance of native gastric cardiac glands: a reduction in the number of layers of subepithelial glands, often with decrease in the size and number of the glands within the lamina propria. There is also a chronic inflammation of the lamina propria with abundant lymphocytes, macrophages, and plasma cells. This evaluation is possible in samples that include the muscularis mucosae, being completely represented in the full thickness of the mucosae. (d) IM tissues, recognized as the presence of goblet cells (special staining [AB-PAS] and immunostaining of MUC2 reveal that they secrete specific mucus), columnar absorptive cells with well-defined brush borders, or Paneth cells. Additionally, IEN refers to morphologically dysplastic epithelium (tortuous glandular structures are lined by mucus-depleted epithelial cells with irregular, elongated, and hyperchromatic nuclei) without breaching the basement membrane.[9]

DNA isolation and bisulfite pyrosequencing

Genomic DNA was isolated from fresh-frozen tissues using the AllPrep DNA/RNA Mini Kit (Cat# 80824, Lot: 166018916, QIAGEN, Germany), according to the manufacturer's protocols. The concentration and purity of the extracted genomic DNA were measured with an ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). Extracted DNA was stored at –80°C until further analysis.

To quantify the methylation levels of candidate cytosine-phosphate-guanosine (CpG) sites in the promoter of EYA4, quantitative bisulfite pyrosequencing was performed. Briefly, 500 ng of total genomic DNA from each of the gastric cardiac tissues was used for bisulfite conversion, using the EZ DNA Methylation Gold Kit (Zymo Research, USA). The primers used were designed using PyroMark Assay Design Software 2.0.2 (QIAGEN) [Supplementary Table S1[Additional file 2]]. One of the PCR primers was biotin labeled. The sequence for analysis is localized to the promoter region of the EYA4 gene. The PCR product was checked by 1% agarose gel electrophoresis (showing one clear band). According to the manufacturer's instructions, the biotinylated PCR product was purified as single-stranded DNA to be used as the template in a pyrosequencing reaction, using the Vacuum Prep Workstation (QIAGEN), and pyrosequencing was then performed using PyroMark Gold Q96 reagent (QIAGEN). The methylation percentage for each CpG site was generated automatically using PyroMark Q96 (QIAGEN), and the results were displayed as a pyrogram with the methylation percentage.

IHC

Immunohistochemical staining was conducted in 38 normal gastric cardiac mucosae, 47 IM tissues, and three IEN tissues. Briefly, the paraffin-embedded tissues were sectioned into 4-μm thickness and placed on adhesion glass slides, followed by baking at 65°C for 1 h. Slides were then deparaffinized in xylene (10 min ×3) and rehydrated with descending concentrations of ethanol (100%, 5 min; 95%, 5 min; 85%, 5 min; and 75%, 5 min) and rinsed with phosphate-buffered saline (PBS). Then, antigen retrieval was performed by heating the slides (immersed in 10 mM sodium citrate buffer, pH = 6.0 [for EYA4] or EDTA, pH = 9.0 [for MUC2]) for 3 min in a pressure cooker. Slides were washed with PBS (3 min ×3), and the endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature, followed by three PBS washes (3 min each). Slides were incubated with EYA4 primary antibody (Cat# ab251675, rabbit polyclonal, Lot: GR3304276-1, Abcam, UK; 1:100 dilution) or MUC2 primary antibody (Cat# ZM-0392, Lot: 21082308, clone Ccp58, ZSGB-Bio, China; ready-to-use) overnight at 4°C. Then, the slides were thoroughly washed in PBS (3 min ×3) and incubated with horseradish peroxidase labeled goat anti-mouse/rabbit secondary antibody (Cat# KIT-5020, Lot: 210224S407c, Maixin Biotechnologies, China) for 30 min at 37°C, followed by an additional three washes in PBS (3 min each). Staining was visualized using the 3,3'-diaminobenzidine (Cat# DAB-0031, Maixin Biotechnologies, China) and counterstained with hematoxylin for 5 min. Finally, slides were dehydrated with series ethanol (75%, 5 min; 85%, 5 min; 95%, 5 min; and 100%, 5 min ×2) and cleared in xyline (5 min ×3). Images were captured on the OLYMPUS B ×53 microscope using Olympus cellSens imaging software (version 1.14).

The protein expression level of EYA4 was estimated semi-quantitatively, based on both staining intensity and proportion of stained cells, according to the following criteria. The intensity of immunostaining was graded from 0 to 3 (0: negative; 1: slightly brown staining; 2: moderately brown staining; and 3: darkly brown staining). The percentage of positively stained cells was graded on a scale of 0–4 (0: negative; 1: 1–10%; 2: 11–50%; 3: 51–80%; and 4: >80%). A final immunostaining score (ranging from 0 to 12) was calculated by multiplying the scores of nuclear staining intensity and percentage of positively stained cells.

Statistical analysis and data visualization

All statistical analyses and data visualization were performed in RStudio (version 2021.09.1.372) (http://www.rstudio.com/) within the R statistical environment (version 4.1.2) (https://www.R-project.org) using the packages dplyr (version 1.0.7), ggplot2 (version 3.3.5), ggrepel (version 0.9.1), factoextra (version 1.0.7), reshape2 (version 1.4.4), pheatmap (version 1.0.12), Gviz (version 1.38.0), GenomicFeatures (version 1.46.1), BSgenome (version 1.62.0), TxDb.Hsapiens.UCSC.hg19.knownGene (version 3.2.2), and stats (version 4.1.2). All continuous variables with non-normal distribution were presented as median with interquartile range (IQR) for the indicated number of biological replicates, and categorical variables were presented as frequency with proportion. Continuous data were assessed for normality using Shapiro–Walk test (shapiro.test R function) before statistical tests of significance were run. Differences in median age and immunostaining score of EYA4 among multiple groups (did not pass normality test; [Figure 1]e and [Figure 3]b) were assessed using the Kruskal–Wallis test, followed by Dunnett's post hoc test for multiple comparisons between groups (R package FSA, version 0.9.1). We used Pearson's Chi-square test (chisq.test R function) to compare the detection rates of gastric cardiac IM in each group (stratified by median age [≤50 vs. >50 years] and sex [female vs. male]. For the methylation rate of candidate CpG sites in normal and IM tissues, a Mann–Whitney U non-parametric test (wilcox.test R function) was used [Figure 2]b. For hierarchical cluster analysis of DNA methylation, z-scaled values were used to calculate Euclidean distance that was applied for clustering using Ward's method [Supplementary Figure S1b]. The statistical tests used in each experiment are described in their respective figure legends. The exact sample size (”n”) in each group, where applicable, was provided in the figures or figure legends. Two-tailed P values < 0.05 were considered statistically significant. Adobe Illustrator CC2015 (Adobe Systems, San Jose, CA) is used to organize figures. The micrographs (such as those in [Figure 3]a) are a magnification of a representative area shown adjacent to them.

Figure 2: Quantitative pyrosequencing analysis reveals DNA methylation of candidate CpG sites in the EYA4 promoter region. (a) schematic diagram showing the genomic location of a 34-bp region (Chr: 133562244-133562277) within EYA4 promoter (indicated by a red vertical bar) and a close-up view of the examined CpG sites (red dashed rectangle) (b) a total of 10 fresh-frozen gastric cardiac tissues (n = 5 for normal and IM tissues, respectively) were used for quantification of DNA methylation levels of five candidate CpG sites (cg11518846, cg20980055, cg20286200, cg05062333, and cg01162672) by pyrosequencing. Box plot showing the median methylation level in normal and IM tissues. Each data dot denotes the average methylation of five candidate CpG sites mentioned above. Indicated P value was calculated by the Mann–Whitney U non-parametric test (c and d) representative pyrograms showing the methylation level of cg01162672, cg05062333, cg20286200, cg20980055, and cg11518846 in normal (c) and IM tissues (d). The percentage of DNA methylation at each CpG site is indicated by shaded areas, and the quality of the result is shown in blue (good quality) or yellow (pass quality). On the x-axis, the nucleotides injected by the pyrosequencer are shown, “E” represents the moment when the enzyme was added to the reaction followed by the substrate “S” that is documented by a small spike. The pyrosequencer added a “T” that is not present in the sequence, thus, no spike was observed. The actual sequence to analyze starts with a “C,” followed by the methylated cytosine here marked with “R.” On the y-axis, luminescence detected by the LCD camera is shown. Source data (b) are provided in [Supplementary Table S3]

Click here to view

Figure 3: EYA4 protein expression in normal and IM tissues. (a) representative images of immunohistochemical staining for EYA4 in normal gastric cardiac tissues and IM tissues. As shown in the upper panel, normal gastric cardiac epithelial cells demonstrate strong nuclear immunostaining for EYA4. The middle panel of immunostaining section of IM tissue highlighting areas of intestinal metaplastic glands (outlined by red dashed line) and normal glands (outlined by blue dashed line). The intestinal metaplastic cells show weak to absent immunostaining compared with normal glandular cells. Prominent loss of EYA4 expression in low-grade intraepithelial neoplasia (IEN) tissue in the context of IM is shown in the lower panel. Scale bars, 50 μm (b) quantitative comparison of immunohistochemical staining for EYA4 in normal (n = 38), IM (n = 47), and IM with IEN (n = 3) tissues. Gray dots represent the immunostaining score for EYA4 in each sample. Statistical significance was analyzed by the Kruskal–Wallis test, followed by Dunnett's post hoc test for multiple comparisons between groups. The upper and lower limits of the box represent 25th and 75th quartile immunostaining score distribution with whiskers extending to 1.5 times the range from top/bottom of the box, the center line within the box corresponds to the median value of the immunostaining score in each group, outliers are not shown. Source data (b) are provided in [Supplementary Table S4]

Click here to view

Results

Higher detection rate of gastric cardiac IM in elder and male participants

The median age of the study participants was 50 (IQR: 42–57) years, there were slightly more female than male participants (50.6% vs. 49.4%). Baseline characteristics of participants are presented in [Table 1]. The most common pathological findings were chronic carditis (494 participants [68.8%; 95% CI: 65.2–72.2]), followed by IM (101 participants [14.1%; 95% CI: 11.7–16.9]) and chronic atrophic carditis (85 participants [11.8%; 95% CI: 9.6–14.5]; [Figure 1]d). Of note, foci of IEN occurred in the context of IM in four (4.0%; 95% CI: 1.3–10.4) of the 101 participants with IM. As shown in [Figure 1]d, the intestinal metaplastic glands are closely packed and lined by irregular cells with elongated and hyperchromatic nuclei.

Table 1: Basic characteristics of the participants enrolled in gastric cardiac biopsy cohort

Click here to view

Among these four histological categories, participants with IM were older than those in other categories (P < 0.001; [Figure 1]e). We then categorized the participants into two age groups using the median age (50 years) of the overall participants in this study as a cutoff. As expected, the detection rates of IM in participants ≤50 years increased from 6.7% (95% CI: 4.5–9.9) to 22.0% (95% CI: 17.8–26.8) in those > 50 years (P < 0.001). Subgroup analysis by sex showed a significantly higher detection rate of gastric cardiac IM in male participants compared to that of female participants (16.9% [95% CI: 13.2–21.3] vs. 11.3% [95% CI: 8.3–15.1], P = 0.04; [Figure 1]f). Individual participant characteristics are detailed in [Supplementary Table S2[Additional file 3]].

Hypermethylation of EYA4 promoter region in gastric cardiac IM tissues

To identify differentially methylated genes in gastric cardiac IM compared with normal gastric cardiac tissue, we reanalyzed a dataset from our group that had originally been generated to assess methylation alterations in gastric cardiac IM.[10] We assigned differentially methylated CpG sites found between normal and IM tissues to candidate genes, which were identified based on a false discovery rate <0.01, the difference in methylation (β value difference) ≥20%, and 19 or more differentially CpG sites located in promoter regions per gene. Through this analysis, we were able to identify five candidate genes showing significant hypermethylation in their promoter regions [Supplementary Figure S1a[Additional file 1]]. Unsupervised clustering analysis using all CpG sites in the candidate genes confirmed an explicit segregation and a clear epigenetic difference between normal and IM tissues [Supplementary Figure S1b]. Among these candidate genes, EYA4 has been reported to be hypermethylated in Barrett's esophagus,[11],[12] we thus, selected EYA4 as a promising biomarker for downstream analysis.

Next, we used a quantitative pyrosequencing assay to assess the methylation status of the five candidate CpG sites for the EYA4 promoter region [Figure 2]a, [Table 2] on an independent set of gastric cardiac samples with sufficient DNA. As shown in [Figure 2]b [Supplementary Table S3[Additional file 4]], IM tissues showed a significant higher DNA methylation level (mean methylation level of five CpG sites) than normal gastric cardiac tissues (P = 0.016), demonstrating the association between gastric cardiac IM and hypermethylation in the EYA4 promoter region. Representative pyrograms for normal and IM tissues are depicted in [Figure 2]c and [Figure 2]d.

Decreased EYA4 protein expression in gastric cardiac IM tissues

The intriguing observation of significant hypermethylation of the EYA4 promoter region in gastric cardiac IM tissues led us to hypothesize that these metaplastic lesions may have lower levels of EYA4 protein compared to the normal gastric cardiac tissues. Formalin-fixed paraffin-embedded biopsy samples were then subjected to immunostaining for EYA4. As shown in [Figure 3]a, EYA4 was uniformly expressed in the nuclei of normal gastric cardiac epithelial cells (upper panel) and its expression was significantly reduced in IM lesions (middle panel). As expected, loss expression of EYA4 was noted in all the three IEN samples included in this study (lower panel). Following image quantification, the immunostaining score of EYA4 was significantly decreased in IM and IEN tissues compared with that in normal gastric cardiac tissues (P < 0.001; [Figure 3]b); [Supplementary Table S4[Additional file 5]]. These results demonstrated that the downregulated expression of EYA4 is a promising biomarker for gastric cardiac IM as well as early gastric cardiac carcinogenesis.

Discussion

It is becoming clear that alterations in DNA methylation are associated with precancerous lesions.[13],[14],[15],[16] However, the role of DNA methylation in gastric cardiac IM has received relatively less attention. Previously, we reported that DNA methylation profiles of gastric cardiac IMs significantly differed from those of normal gastric cardiac mucosa. In this study, we researched the methylation status of the EYA4 promoter region and gained insights on epigenetic markers in gastric cardiac IM. In our earlier study, obvious hypermethylation of gene promoter regions was shown in gastric cardiac IM samples. By conducting pyrosequencing in the candidate CpG sites of EYA4 and IHC analysis of EYA4 protein, our present study revealed that gastric cardiac IM lesions exhibited hypermethylation in the promoter of EYA4 and showed a reduced protein expression. This work provides evidence for promoter hypermethylation of EYA4 as a promising epigenetic biomarker in gastric cardiac IM.

To date, the data regarding the detection rate of gastric cardiac IM in cancer-free individuals are limited. In this study, we assessed the pathology of the gastric cardiac mucosae covering more than 700 cancer-free individuals in Guangdong Province, China. We specifically focused on the detection rate of gastric cardiac IM to provide baseline data for further investigation of this premalignant lesion. Based on pathological assessment, the overall gastric cardiac IM detection rate in our study population was 14.1% (95% CI: 11.7–16.9). However, there are wide variations in the detection rates of gastric IMs among different studies,[17],[18] partly because of the unavoidable sampling bias, because most IMs showed only focal involvement. It deserves to be noted that in our study, the detection rates for gastric cardiac IM increased with age and were higher for men than for women, as shown in [Figure 1]f. These findings were consistent with that of previous studies on gastric IMs.[19],[20] Most likely, this age-dependent rise reflects the natural history of gastric cardiac IM and can be explained by the long duration of exposure to environmental risk factors. Therefore, it is necessary to consider the age and sex for future screening and surveillance for premalignant lesions of gastric cardia. Indeed, not all patients with gastric cardiac IMs will progress to cancer ultimately. Of note, we found IEN in the context of IM in four subjects (4/101; 4.0%) of the study participants. Thus, further work should be done to identify a subset of IM subjects at high risk of neoplastic progression.

DNA methylation changes have been studied in multiple cancer types.[21],[22],[23],[24] Furthermore, increasing evidence shows that aberrant DNA methylation events occur even in samples of precancerous lesions.[25] Thus, such epigenetic events are poised to become ideal biomarkers for early stage cancer.[26] However, only a few studies have focused on DNA methylation biomarkers in gastric non-cardiac IMs,[15],[27],[28] and even less on IMs arising from gastric cardia. We recently conducted array-based genome-wide DNA methylation analysis in gastric cardiac IMs compared with normal gastric cardiac mucosae[10] and found that EYA4 gene was significantly hypermethylated in gastric cardiac IMs. In this study, we further confirmed that hypermethylation of the EYA4 gene promoter was evident in gastric cardiac IMs [Figure 2]. This aligns with a previous report highlighting a link between EYA4 promoter hypermethylation and Barrett's esophagus, a condition of IM in the distal esophagus.[12] Furthermore, several studies reported the hypermethylation of EYA4 in different cancer types.[29],[30],[31] These findings support the claim that promoter DNA methylation of EYA4 is closely associated with IM development and is a very early event during multistage gastric cardiac carcinogenesis. Therefore, it will be worth investigating how well an EYA4 methylation test can detect dysplastic and cancerous lesions in future studies.

DNA hypermethylation plays an important role in carcinogenesis because it could cause the silencing of some pivotal genes, especially tumor suppressor genes.[32],[33] In this study, we observed that EYA4 protein was significantly reduced or not present in gastric cardiac IMs and dysplastic lesions, compared with normal tissues [Figure 3]a, supporting our hypothesis that EYA4 expression reduction might be, at least partly, because of promoter DNA hypermethylation. These data indicate that EYA4 is a promising biomarker for gastric cardiac IMs. EYA4 is one of the four members of EYA gene family that was initially identified in Drosophila.[34] Of note, interesting clues about the role of EYA4 protein in tumorigenesis are emerging rapidly, namely, its tumor suppressor role in esophageal squamous cell carcinoma,[31] colorectal carcinoma,[35] pancreatic adenocarcinoma,[36] hepatocellular carcinoma,[37] and bladder cancer.[38] Thus, it is possible that reduced expression of EYA4 protein contributes to IM development and is an early molecular event during gastric cardiac carcinogenesis. Future investigations should aim to elucidate the functional role and the exact mechanism of EYA4 in IM development.

The major limitation of this study lies in the small sample size for pyrosequencing validation, for the methylation status of EYA4 promoter. Therefore, continued efforts to investigate the frequency of EYA4 methylation and its functional relevance, will improve our understanding of the role of EYA4 in gastric cardiac IM. Another limitation of the study is the small sample size of IEN (n = 3) used for IHC.

In summary, the promoter hypermethylation of EYA4 may contribute to the downregulation of protein expression in gastric cardiac IM, a precursor lesion of gastric cardiac cancer. This finding highlights the important role of aberrant DNA methylation in the EYA4 promoter region in the pathogenesis of gastric cardiac IM [Figure 4].

Figure 4: Schematic model depicting the role of hypermethylation in the EYA4 promoter region in gastric cardiac IM development. Hypermethylation-related reduced expression of the EYA4 protein may contribute to the development of gastric cardiac IM and appears to be a promising biomarker for this premalignant lesion

Click here to view

Financial support and sponsorship

This study was supported by Project of Educational Commission of Guangdong Province of China (Grant No. 2017KQNCX068), the research grants from the Shantou Science and Technology Bureau (Grant No. 210712186880511; Grant No. 220507236491772), the Open Fund of Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology (Grant No. GDKL202209), Medical Scientific Research Foundation of Guangdong Province of China (Grant No. A2022329), and the Huizhou Science and Technology Bureau (Grant No. 2021WC0106069).

Conflicts of interest

There are no conflicts of interest.

References

1.Fan J, Li J, Guo S, Tao C, Zhang H, Wang W, et al. Genome-wide DNA methylation profiles of low- and high-grade adenoma reveals potential biomarkers for early detection of colorectal carcinoma. Clin Epigenetics 2020;12:56.

2.den Hoed CM, Holster IL, Capelle LG, de Vries AC, den Hartog B, Ter Borg F, et al. Follow-up of premalignant lesions in patients at risk for progression to gastric cancer. Endoscopy 2013;45:249-56.

3.Kim N, Park RY, Cho SI, Lim SH, Lee KH, Lee W, et al. Helicobacter pylori infection and development of gastric cancer in Korea: Long-term follow-up. J Clin Gastroenterol 2008;42:448-54.

4.Whiting JL, Sigurdsson A, Rowlands DC, Hallissey MT, Fielding JW.The long term results of endoscopic surveillance of premalignant gastric lesions. Gut 2002;50:378-81.

5.Morris MR, Latif F. The epigenetic landscape of renal cancer. Nat Rev Nephrol 2017;13:47-60.

6.Saghafinia S, Mina M, Riggi N, Hanahan D, Ciriello G. Pan-cancer landscape of aberrant DNA methylation across human tumors. Cell Rep 2018;25:1066-80.e8.

7.Natale F, Vivo M, Falco G, Angrisano T. Deciphering DNA methylation signatures of pancreatic cancer and pancreatitis. Clin Epigenetics 2019;11:132.

8.Rosai J.Rosai and Ackerman's Surgical Pathology e-book. Elsevier Health Sciences; 2011.

9.Rugge M, Correa P, Dixon MF, Hattori T, Leandro G, Lewin K, et al. Gastric dysplasia: The Padova international classification. Am J Surg Pathol 2000;24:167-76.

10.Lin R, Li C, Liu Z, Wu R, Lu J. Genome-wide DNA methylation profiling identifies epigenetic signatures of gastric cardiac intestinal metaplasia. J Transl Med 2020;18:292.

11.Kaz AM, Wong CJ, Luo Y, Virgin JB, Washington MK, Willis JE, et al. DNA methylation profiling in Barrett's esophagus and esophageal adenocarcinoma reveals unique methylation signatures and molecular subclasses. Epigenetics 2011;6:1403-12.

12.Zou H, Osborn NK, Harrington JJ, Klatt KK, Molina JR, Burgart LJ, et al. Frequent methylation of eyes absent 4 gene in Barrett's esophagus and esophageal adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2005;14:830-4.

13.Schmitz M, Eichelkraut K, Schmidt D, Zeiser I, Hilal Z, Tettenborn Z, et al. Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages. BMC Cancer 2018;18:1197.

14.Moinova HR, LaFramboise T, Lutterbaugh JD, Chandar AK, Dumot J, Faulx A, et al. Identifying DNA methylation biomarkers for non-endoscopic detection of Barrett's esophagus. Sci Transl Med 2018;10:eaao5848. doi: 10.1126/scitranslmed.aao5848.

15.Huang KK, Ramnarayanan K, Zhu F, Srivastava S, Xu C, Tan ALK, et al.Genomic and epigenomic profiling of high-risk intestinal metaplasia reveals molecular determinants of progression to gastric cancer. Cancer Cell 2018;33:137-50.e5.

16.Oh TJ, Oh HI, Seo YY, Jeong D, Kim C, Kang HW, et al. Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer. Clin Epigenetics 2017;9:126.

17.Da B, Jani N, Gupta N, Jayaram P, Kankotia R, Yao Yu C, et al. High-risk symptoms do not predict gastric cancer precursors. Helicobacter 2019;24:e12548.

18.Kang KP, Lee HS, Kim N, Kang HM, Park YS, Lee DH, et al. Role of intestinal metaplasia subtyping in the risk of gastric cancer in Korea. J Gastroenterol Hepatol 2009;24:140-8.

19.McNamara D, Buckley M, Crotty P, Hall W, O'Sullivan M, O'Morain C.Carditis: All Helicobacter pylori or is there a role for gastro-oesophageal reflux?Scand J Gastroenterol 2002;37:772-7.

20.Felley C, Bouzourene H, VanMelle MB, Hadengue A, Michetti P, Dorta G, et al. Age, smoking and overweight contribute to the development of intestinal metaplasia of the cardia. World J Gastroenterol 2012;18:2076-83.

21.Baylin SB, Jones PA.A decade of exploring the cancer epigenome-biological and translational implications. Nat Rev Cancer 2011;11:726-34.

22.Zhao SG, Chen WS, Li H, Foye A, Zhang M, Sjöström M, et al. The DNA methylation landscape of advanced prostate cancer. Nat Genet 2020;52:778-89.

23.Klughammer J, Kiesel B, Roetzer T, Fortelny N, Nemc A, Nenning KH, et al. The DNA methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space. Nat Med 2018;24:1611-24.

24.Hovestadt V, Jones DT, Picelli S, Wang W, Kool M, Northcott PA, et al.Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing. Nature 2014;510:537-41.

25.Peng DF, Kanai Y, Sawada M, Ushijima S, Hiraoka N, Kitazawa S, et al. DNA methylation of multiple tumor-related genes in association with overexpression of DNA methyltransferase 1 (DNMT1) during multistage carcinogenesis of the pancreas. Carcinogenesis 2006;27:1160-8.

26.Dor Y, Cedar H. Principles of DNA methylation and their implications for biology and medicine. Lancet 2018;392:777-86.

27.Shin CM, Kim N, Lee HS, Park JH, Ahn S, Kang GH, et al. Changes in aberrant DNA methylation after Helicobacter pylori eradication: A long-term follow-up study. Int J Cancer 2013;133:2034-42.

28.Sugimoto R, Habano W, Yanagawa N, Akasaka R, Toya Y, Sasaki A, et al. Molecular alterations in gastric cancer and the surrounding intestinal metaplastic mucosa: An analysis of isolated glands. Gastric Cancer 2021;24:382-91.

29.Emmett RA, Davidson KL, Gould NJ, Arasaradnam RP.DNA methylation patterns in ulcerative colitis-associated cancer: A systematic review. Epigenomics 2017;9:1029-42.

30.Hou X, Peng JX, Hao XY, Cai JP, Liang LJ, Zhai JM, et al. DNA methylation profiling identifies EYA4 gene as a prognostic molecular marker in hepatocellular carcinoma. Ann Surg Oncol 2014;21:3891-9.

31.Luo M, Li Y, Shi X, Yang W, Zhou F, Sun N, et al. Aberrant methylation of EYA4 promotes epithelial-mesenchymal transition in esophageal squamous cell carcinoma. Cancer Sci 2018;109:1811-24.

32.Sharma G, Mirza S, Parshad R, Srivastava A, Gupta SD, Pandya P, et al. Clinical significance of promoter hypermethylation of DNA repair genes in tumor and serum DNA in invasive ductal breast carcinoma patients. Life Sci 2010;87:83-91.

33.Jones PA. Functions of DNA methylation: Islands, start sites, gene bodies and beyond. Nat Rev Genet 2012;13:484-92.

34.Borsani G, DeGrandi A, Ballabio A, Bulfone A, Bernard L, Banfi S, et al. EYA4, a novel vertebrate gene related to Drosophila eyes absent. Hum Mol Genet 1999;8:11-23.

35.Kim SJ, Tae CH, Hong SN, Min BH, Chang DK, Rhee PL, et al. EYA4 acts as a new tumor suppressor gene in colorectal cancer. Mol Carcinog 2015;54:1748-57.

36.Mo SJ, Liu X, Hao XY, Chen W, Zhang KS, Cai JP, et al. EYA4 functions as tumor suppressor gene and prognostic marker in pancreatic ductal adenocarcinoma through β-catenin/ID2 pathway. Cancer Lett 2016;380:403-12.

37.Zhu XX, Li JH, Cai JP, Hou X, Huang CS, Huang XT, et al. EYA4 inhibits hepatocellular carcinoma by repressing MYCBP by dephosphorylating β-catenin at Ser552. Cancer Sci 2019;110:3110-21.

38.Dong W, Bi J, Liu H, Yan D, He Q, Zhou Q, et al. Circular RNA ACVR2A suppresses bladder cancer cells proliferation and metastasis through miR-626/EYA4 axis. Mol Cancer 2019;18:95.