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Gastric cancer (GC) is the third highest cause of global cancer mortality.1 It is a heterogeneous disease with multiple environmental etiologies and alternative carcinogenic pathways.2, 3, 4, 5, 6 The development of noninvasive biomarkers to detect early GC (EGC) and/or reflect an individual’s cancer risk is essential to reduce GC mortality.7, 8 Among the various methods for detection of genetic and epigenetic alterations,6 DNA methylation is more appropriate than mutations for molecular detection of GC. Previous strategies have relied on the comparison of methylation levels between tumor and adjacent nontumor sites, to find genes that are specifically methylated in cancer. However, discovery of tumor-specific hypermethylated genes using this strategy is challenging, because chronic inflammation of the gastric mucosae (mainly due to H. pylori infection and aging) also induces aberrant methylation.9, 10, 11, 12, 13, 14 Moreover, the process relies on endoscopic biopsy, which is a topical procedure that only samples a small portion of tissue.15 As a result of the uneven distribution of the atrophy or intestinal metaplasia, this restricted biopsy can lead to flawed evaluation of methylation status.16 In light of these problems, we have developed a method that uses gastric wash-derived DNA for EGC detection and have employed it to detect methylation of genes such as MINT25 (ref. 15). Gastric washes contain large amounts of DNA recovered from cells on the surface of the stomach, making it simple to collect DNAs from patients endoscopically, both before and after endoscopic resection (ER). Comparing DNA methylation levels in each of the patients before and after treatment is useful for the identification of genes that are specifically methylated in EGC; this process is not biased by factors such as aging, chronic inflammation or H. pylori infection. On the other hand, it is possible that GC is subject to field effects, and therefore markers that are not altered by the ER could still play a role in its diagnosis. In this regard, several markers such as miR34b/c and miR-124a-3 (ref. 13) have been already reported. As minimal invasive treatment is widely used for EGC patients, the identification of appropriate markers for detection of residual tumors after non-curative ER is critical.17, 18 One of our main objectives is to develop markers that could be useful for detection of tumors that remain after non-curative ER and/or those that recur after curative ER in EGC patients. Therefore, in the present study we focused on markers that exhibit quantitative changes following ER. We previously performed methylated CpG island amplification microarray (MCAM) analysis using 12 gastric washes (6 before and 6 after ER in each of the same patients).19 The 18 probes (total 36,579) corresponding to 11 unique genes (total 9,021) were selected as candidate tumor-specific methylated genes after calculations were made using Gene-Spring GX (Agilent Technologies, Santa Clara, CA) software based on the DNA methylation intensity measurements. Among these genes, we have shown that the silencing of sex determining region Y-Box 17 (SOX17) occurs frequently in EGC and may have a key role in the development and progression of the disease. A number of genes are differentially methylated in GC and noncancer tissues following H. pylori infection. On the other hand, H. pylori eradication decreases aberrant DNA methylation in a gene-specific manner. These issues complicate the successful development of a methylation analysis pipeline in GC. Although there has been a striking decrease in the prevalence of H. pylori infection, especially in younger populations, it is important to identify biomarkers for early detection of GC that are not affected by either H. pylori infection or history of eradication. In this regard, SOX17 methylation is not ideal, because levels of SOX17 methylation were significantly affected by H. pylori infection in gastritis samples. The BarH-like 2 homeobox protein (BARHL2) gene is a candidate H. pylori-independent biomarker. This is because 6 of the forementioned 18 probes methylated in EGC analyzed by MCAM corresponded this gene and BARHL2 was one of the most significantly altered genes based on the Cy5/Cy3 (pre-ER/post-ER) signal in MCAM. A pilot study showed that BARHL2 methylation may not be affected by H. pylori infection. Although it is not known whether BARHL2 has a role in human malignancy, the function of BarH family of homeodomain proteins as transcriptional regulators has an impact on cell fate specification, cell differentiation, migration, and survival.20, 21 Together, these data indicate that BARHL2 inactivation may play a role in GC. In this study, we analyzed epigenetic alterations of BARHL2, as well as the role of the BARHL2 protein in GC cell biology. We also determined whether analysis of BARHL2 methylation using gastric wash-derived DNA and/or gastric juice-derived exosomal DNA (exoDNA) could be applied for the detection of EGC. Here we demonstrate that silencing of BARHL2 was correlated with hypermethylation of its promoter in GC cell lines. Treatment of these cell lines with 5-aza-dC restored BARHL2 expression. Moreover, combined treatment of 5-aza-dC and TSA synergized to restore BARHL2 expression, indicating that cytosine methylation and histone deacetylation have a role in silencing of this gene in GC. The synergistic role was more evident in the MKN45 cell line than in MKN7 cells. This may be because patterns of methylation, histone acetylation, and the expression of other transcription factors involved in the regulation of BARHL2 transcription are different between the cell lines. In this regard, increased histone acetylation may be associated with increased BARHL2 expression in BARHL2 methylation-negative NUGC3 cells treated with TSA. We examined BARHL2 methylation status in gastric wash-derived DNA samples obtained from control samples and EGC patients before and after ER. We found that levels of BARHL2 methylation as determined from gastric washes and formalin-fixed paraffin-embedded samples were well correlated, supporting the notion that gastric washes reflect biopsy results at the DNA methylation level.15 There was a correlation between BARHL2 methylation status and gene expression in EGC samples, suggesting that BARHL2 methylation is functionally significant. Interestingly, BARHL2 methylation was low in 18 normal and 14 gastritis samples, irrespective of H. pylori infection. As for EGC samples before ER, there was no significant difference in BARHL2 methylation levels between EGC samples with and without H. pylori infection. We note that clinical tests for H. pylori infection detect only the current (culture and urease tests) or recent (serum antibody test) status of H. pylori infection and cannot detect past exposure to H. pylori.9, 23, 24 Nevertheless, these results suggest that BARHL2 methylation is a tumor-specific event that is not influenced by atrophy of the gastric mucosa or H. pylori infection that may accompany gastric carcinogenesis. BARHL2 methylation levels were significantly higher in EGC samples before ER than they were in control samples. Following curative ER, BARHL2 methylation levels significantly decreased to levels of controls in most patients. Therefore, gastric wash-based BARHL2 methylation analysis could be useful for early detection of remaining tumors after non-curative ER and/or recurrence after curative ER in EGC patients. Nevertheless, an endoscopy is necessary to obtain gastric washes. Exosomes in the gastric juice may provide an alternative to gastric washes that can be used for GC molecular diagnostics.22 Using exoDNA derived from gastric juice, we were able to detect BARHL2 methylation, which reflects the nuclear DNA methylation status of the corresponding tumor. When the cutoff value for BARHL2 methylation was 20%, sensitivity was 90% and specificity was 100%. BARHL2 methylation was detected in both early and advanced GC of intestinal and diffuse types. These findings suggest that BARHL2 methylation analysis of exoDNA derived from gastric juice has utility as a biomarker for detection of both early and advanced GC. The high specificity of the approach is supported by the fact that BARHL2 methylation is not influenced by atrophy of the gastric mucosa or H. pylori infection. Combined with its high methylation frequency, these properties make BARHL2 methylation an excellent candidate for future diagnostic applications for the early detection of GC. The results of methylation analyses were supported by our results with immunohistochemistry. BARHL2 expression was frequently downregulated in GC tissues, irrespective of tumor grading or stage, but was preserved in gastric epithelial cells, as well as those from patients with gastritis or adenoma. These results further support the notion that BARHL2 silencing is a tumor-specific event in GC. High levels of BARHL2 methylation were detected in only three of seven GC cell lines. BARHL2 was selected as a marker based on the MCAM data of six test set samples (well or moderately differentiated cancer). Importantly, BARHL2 methylation was detected in not only well-differentiated but also poorly differentiated GC cell lines. Considering the data of clinical samples, therefore, the relatively low frequency of BARHL2 methylation in GC cell lines does not necessarily discourage the role of BARHL2 methylation as a diagnostic tool. Given the silencing of BARHL2 we observed, we hypothesized that restoration of expression might suppress GC cell growth. This was indeed the case in both MKN7 and MKN45 cell lines, although the cell cycle phases in these cells were not affected by BARHL2. We also observed that BARHL2 induced expression of several transcription factors involved in cell differentiation in MKN45 cells but not in MKN7 cells. We suggest that this is because MKN7 is already a well-differentiated GC line, whereas MKN45 is poorly differentiated. The BarH family of homeodomain proteins has essential roles in cell fate specification, cell differentiation, migration, and survival through transcriptional regulation.20, 21 Therefore, BARHL2 may regulate gastric epithelial cell features through modulation of the network of transcriptional factors and its inactivation may have a role in GC. As gastrointestinal endoscopy is costly and painful for patients, it is difficult to incorporate the technique into routine clinical settings, especially in developing countries.22 In contrast, gastric juice samples can be obtained easily and repeatedly, and exoDNA is not easily denatured by gastric acidity.22 Although our data require further validation, detection of BARHL2 methylation in gastric washes and/or gastric juice-derived exosomes may be a novel and less invasive tool for EGC detection.  Guarantor of the article: Hiroyuki Yamamoto, MD, PhD. Specific author contributions: Study design: H. Yamamoto and Y. Watanabe. Sample preparation: Y. Watanabe, R. Oikawa, R. Morita, Y. Yoshida, and T. Maehata. Data analysis and interpretation: H. Yamamoto, Y. Watanabe, R. Oikawa, R. Morita, Y. Yoshida, and T. Maehata. Manuscript preparation: H. Yamamoto, Y. Watanabe, H. Yasuda, and F. Itoh. All the above-mentioned authors have approved the final draft submitted. Financial support: This work was supported in part by a grants-in-aid of The Public Trust For Clinical Cancer Research (to HY), Suzuken Memorial Foundation (to FI), Daiwa Securities Health Foundation (to FI), The Japanese Foundation for Research and Promotion of Endoscopy (to HY), and Kobayashi Foundation for Cancer Research (to HY). Potential competing interests: None.

clinical and translational gastroenterology

BARHL2 Methylation Using Gastric Wash DNA or Gastric Juice Exosomal DNA is a Useful Marker For Early Detection of Gastric Cancer in an H. pylori -Independent Manner