This is the first study to report the molecular alterations associated with metachronous lesions developing on scars after curable resection by ESD for EGC. EGC with metachronous lesions developing on scars was detected in only 0.5% of all ESD cases. The median duration for new atypical lesions developing on scars after curable ESD was 22 months (range, 14–49 months). EGC with metachronous lesions developing on scars was compared to EGC with no confirmed evidence of metachronous lesions at > 3 years after curable ESD (controls), and we identified no clear clinicopathological features between the two groups. Furthermore, H. pylori-negative EGC patients with non-atrophic mucosa differed from controls in several aspects, including age, histological type, macroscopic type, and H. pylori status. Therefore, EGC with metachronous lesions developing on scars after curable ESD might have formed as a result of molecular changes in the background atrophic mucosa, and such changes may play a critical role in carcinogenesis among epigenetic factors.
The carcinogenic process in the gastric mucosa with atrophy is mainly attributable to field epigenetic abnormalities. Atrophy related to old age and H. pylori is known to be caused by irritation due to persistent chronic inflammation.[10, 31] Chronic inflammation leads to DNA and epigenetic abnormalities in the gastric mucosa.[11, 32] In particular, epigenetic alterations involved in gastric carcinogenesis are considered to contribute to a two-fold higher risk than that of esophageal squamous cell carcinoma, which may be due to the so-called “field cancerization,” in which epigenetic changes responsible for gene expression have already occurred in the background gastric mucosa. EGC with metachronous lesions developing on scars after curable ESD was pathologically confirmed after curative resection by ESD and was not caused by residual cancer. Nevertheless, considering the newly developed cancer in the same area, it is strongly suggested that the TAM already harbored cancer-like changes. We focused our attention on CDO1, a hypermethylated gene with particularly high specificity in human cancers, because CDO1 is the most promising candidate gene for cancer-specific epigenetic changes.
Among the cancer-specific methylation genes in gastric cancer, CDO1 has the highest AUC (0.95), which distinguishes cancerous from noncancerous tissues. This finding suggests that CDO1 methylation in cancer cells may be strongly associated with carcinogenesis. The current study revealed that the TAM in EGC was hypermethylated, albeit not as much as the tumor (Fig. 1-A). Our previous study on CDO1 methylation in PDAC showed that 90% of cancerous tissues were methylated, whereas non-CP exhibited no methylation. Additionally, in remnant gastric cancer, CDO1 was more highly methylated in cancerous tissue than in the noncancerous mucosa, which is far from the tumor; however, its methylation level was clearly detected in the noncancerous mucosa. Furthermore, CDO1 methylation has been reported to be significantly higher in precancerous lesions of other cancers such as small bowel cancer, colorectal cancer, and intraductal papillary mucinous neoplasm than in noncancerous tissues.[24, 25, 35] In our study, for the first time, we demonstrated that CDO1 hypermethylation in the TAM was more frequently detected in EGC with metachronous lesions developing on scars than in EGC with no evidence of metachronous lesions. Moreover, using the optimal CDO1 methylation value (43.4), we identified as many as nine out of 11 cases of EGC with metachronous lesions developing on scars after curable ESD. Therefore, our study confirmed that the TAM in EGC with metachronous lesions developing on scars exhibited cancer patterns.
Since EGC with metachronous lesions developing on scars after curable ESD is extremely rare, even if this prediction is possible, it may not have a significant clinical impact. Nonetheless, this is considered extremely useful as a model to investigate the carcinogenesis of gastric cancer. In our study, we believe that this is a very important finding, as the result of EGC with metachronous lesions developing on scars reflects epigenetic abnormalities that had already occurred. Regarding prediction of EGC, Asada et al. examined gene methylation at a fixed point in the antrum (the lesser curvature at 2 cm from the pyloric ring) and reported a 2.3-fold increased risk of heterogeneous gastric cancer. However, with respect to the prediction of EGC with metachronous lesions developing on non-scar tissue, identifying the site of development with gastric cancer is difficult, and the site of development cannot be accurately determined by prior biopsy. In contrast, measurement of CDO1 methylation in ESD tissue for the T and the TAM may predict the occurrence of new cancers in scars after treatment. For this reason, metachronous lesions developing on scars after curable ESD may be an important design model for predicting the development of EGC.
Surprisingly, EGC cases without atrophy (N group) showed no CDO1 methylation in the TAM or even in the tumor. As the age of carcinogenesis in these cases is younger than that in conventional gastric cancer, there exists the possibility that de novo carcinogenesis is more likely to generate such a specific gastric cancer than epigenetic carcinogenesis. In our study, the histological types of H. pylori-negative EGC with no atrophic mucosa (N group) were all signet-ring cells, whereas advanced tumors with signet-ring cell histology exhibited CDO1 hypermethylation, similar to other histological types. This finding suggests that CDO1 methylation does not occur in EGC with signet-ring histology that remains within the mucosa. To more accurately determine this, further pathological analysis of CDO1 hypermethylation is necessary.
Our study has several important limitations. We analyzed the methylation of a single gene, CDO1. DNA methylation is not a change that occurs in only a single gene; there is a strong association between methylated genes. We have recently reported that when combined with HOPX/Reprimo/CDH1, CDO1 methylation can predict future occurrences of remnant gastric cancer. Therefore, other methylated genes should be considered in combination with CDO1 in order to develop a more accurate system for prediction and clinical application. Furthermore, in the present study, using EGCs with metachronous lesions were not identified more than 3 years after curable ESD as a control, the optimal methylation value of CDO1 (43.4) was used to identify 9 of 11 cases of EGC with metachronous lesions developing on the scar after curable ESD. However, among the EGC in which metachronous lesions were not identified at least more than 3 years after ESD, we found seven cases with high CDO1 methylation of the TAM. These cases need to be closely monitored owing to the possibility of new lesions development on the scar.
In conclusion, CDO1 promoter DNA methylation may be an important biomarker for predicting EGC with metachronous lesions developing on scars after curable ESD. If an accurate system can be developed to predict EGC with metachronous lesions developing on scars after curable ESD in the near future, molecular information on lesions after radical ESD will be a new index to propose the most optimal follow-up endoscopy program after ESD treatment.