BACKGROUND: No current biomarkers are available for clear cell carcinoma of the kidney (RCC). Many studies have looked at differential gene expression in kidney cancer and most have concentrated on the genes that are up-regulated in RCC. Our strategy is to concentrate on the down-regulated genes and looked for epigenetic changes within the DNA of these genes. We then correlated these DNA/RNA changes at the protein level so that potential diagnostics can be developed at multiple levels.
METHODS: We previously reported microarray gene expression of nine patient specimens. An analysis of this data using novel bioinformatics tools found 158 genes that were significantly differentially expressed. Of the 158 genes, we chose 19 down-regulated genes that had CpG rich islands within their DNA sequence. Methylation patterns were analyzed across pairs of histologically normal and RCC samples from 38 individuals using a high-throughput MALDI-TOF mass spectroscopy analysis. Significantly hypermethylated genes were determined through Binomial and Wilcoxon signed-rank tests. We validated these findings at the RNA level with QRT-PCR, Western Blotting, and immunohistochemistry.
RESULTS: We found 7 significantly over-methylated regions (p<0.05) from 6 down-regulated genes, all of which are reported for the first times to be hypermethylated in RCC. Three were peviously reported to be candidate tumor suppressor genes in different tumor types. Two of the three were previously implicated in RCC but not investigated at an epigenetic level. QRT-PCR validated the significant gene downregulation (p<0.05) in 5 of 6 genes while Western Blotting revealed significant protein down-regulation in 4 of 5 genes (p<0.05). Immunohistochemistry revealed less protein in the clear cell cancers versus the normal kidney tissue in all 5 genes.
CONCLUSIONS: The detection of tissue hypermethylation in these highly down-regualted genes suggest that assaying for their methylation status using cells or cell free nucleic acids from urine or blood could provide the basis for a viable diagnostic that can be rapid and inexpensive.