P-0198 Identification of Prognostic Methylation Markers in Patients With Early Stage Colorectal Cancer

ABSTRACT Introduction Colorectal cancer (CRC) is one of the most common forms of cancer. Surgical excision at clinical stages I and II is highly effective, but in about 20-30% of patients, the disease can recur. Therefore, it is highly important to identify patients at high risk for disease recurrence, who would benefit from adjuvant chemotherapy. Aberrant promoter methylation represents a hallmark of colorectal cancer; therefore detection of promoter methylation could be a suitable marker to identify such high risk patients. The overall objective of this study was to identify prognostic methylation markers to assess prognosis in stage II CRC patients. Methods For selection of candidate methylated genes in cancer patients we performed a Methyl Profiler Assay (Qiagen) of 48 gene promoters in two different cell lines, the tumorigenic cell line SW480 and the non-tumorigenic cell line Caco-2. To assess whether promoter methylation was associated with transcriptional silencing, we analyzed mRNA expression in the presence or absence of the demethylating agent 5-Aza-2′-dCytidine (5-Aza) in 4 different colon cancer cell lines (SW480, HT29, HCT116, Caco-2). Additionally, we analyzed the methylation status using established MethyLight assays after 5-Aza treatment compared to the untreated control. Markers that showed promoter demethylation associated with an increase in expression (>10-fold) were then analyzed in a set of 57 primary tumors from stage II CRC patients. Results Of 48 gene promoters 19 showed aberrant methylation in SW480 while 16 promoters where differentially methylated in Caco-2. To verify that the methylation is leading to down-regulation of these genes, we chose 17 of the methylated markers for qPCR analysis after treatment with the demethylating 5-Aza in 4 colorectal cancer cell lines. In all cell lines expression of WIF1 could be restored after 5-Aza treatment. Three cell lines showed also a significant increase in expression of UHCL1. Caco-2 did not show an increase in UHCL1 expression which is in line with results of the Methyl Profiler and the MethyLight assay where the UCHL1 promoter did not reveal any aberrant methylation. For all other cell lines we detected a significant decrease of DNA methylation after 5-Aza treatment corresponding to the mRNA analysis. To evaluate whether this aberrant methylation is also present in clinical samples we analyzed a set of 57 primary tumors from stage II CRC patients. Of 57 tumors 13 (22.8%) could not be analyzed due to low DNA concentrations. The WIF1 promoter was aberrantly methylated in 59.6% (35 of 57) whereas UCHL1 promoter hypermethylation was seen in 29.8% of the tumors (17 of 57). Conclusion Taken together, these results indicate a direct association between WIF1 and UCHL1 promoter methylation and repression of expression. Aberrant promoter methylation of WIF1 and UCHL1 was found in 59.6% and 29.8% of primary tumors, respectively. Thus, WIF1 and UCHL1 appear to be suitable for further analysis and prognostic evaluation of progression and recurrence of CRC.