GFRA3 promoter methylation may be associated with decreased postoperative survival in gastric cancer

Abstract

Background: A large number of epigenetic alterations has been found to be implicated in the etiology of gastric cancer. We have studied the DNA methylation status of 27 500 gene promoter regions in 24 gastric adenocarcinomas from a Norwegian cohort, and aimed at identifying the hypermethylated regions. We have compared our findings to the gene expression in the same tissue, and linked our results to prognosis and survival.

Methods: Biopsies from gastric adenocarcinomas and adjacent normal gastric mucosa were obtained from 24 patients following surgical resection of the tumor. Genome-wide DNA methylation profiling of the tumor and matched non-cancerous mucosa was performed. The results were compared to whole transcriptome cDNA microarray analysis of the same material.

Results: Most of the gene promoter regions in both types of tissue showed a low degree of methylation, however there was a small, but significant hypermethylation of the tumors. Hierarchical clustering showed separate grouping of the tumor and normal tissue. Hypermethylation of the promoter region of the GFRA3 gene showed a strong correlation to post-operative survival and several of the clinicopathological parameters, however no difference was found between the two main histological types of gastric cancer. There was only a modest correlation between the DNA methylation status and gene expression.

Conclusions: The different DNA methylation clusters of the tumors and normal tissue indicate that aberrant DNA methylation is a distinct feature of gastric cancer, although there is little difference in the overall, and low, methylation levels between the two tissue types. The GFRA3 promoter region showed marked hypermethylation in almost all tumors, and its correlation with survival and other clinicopathological parameters may have important prognostic significance.

Keywords: DNA methylation; GFRA3; Gastric cancer; Gene expression; Prognosis; Survival.

Fig. 1

Histogram of the ∆β values. The frequency of the ∆β values in the dataset of >27 000 CpG sites shows that most CpG sites demonstrated little variance between gastric tumor and normal tissue

Fig. 2

A heatmap presentation of the DNA methylation levels of the entire dataset of 27564 CpG sites in 24 tumor and normal gastric cancer tissue pairs. A value of 0.00 (most green) indicates fully unmethylated, whereas a value of 1.00 (most red) indicates entirely methylated locus. The figure demonstrates that the majority of CpG sites in the dataset, including both tumor and normal tissue, showed low levels of DNA methylation

Fig. 3

Hierarchical clustering of the normal and tumor samples. Top row: Tumor samples are shaded grey, normal gastric tissue samples are white. A value of 0.00 (most green) indicates fully unmethylated, whereas a value of 1.00 (most red) indicates entirely methylated locus. Most normal tissue samples seem to concentrate on the left side of the heatmap, whereas most tumor samples aggregate on the right, indicating similarities within the two groups

Fig. 4

Kaplan Mayer survival plot of patients with resected gastric tumors. High and low methylated GFRA3 groups were constructed, using the mean value as the group divider. Individuals with hypermethylation of the GFRA3 promoter region showed a highly unfavorable prognosis, whereas individuals with a low degree of methylation at that locus demonstrated a relatively good prognosis