Abstract 1049: Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes using anion exchange high performance liquid chromatography
Authors: Yotani Takuya , Yamada Yuriko , Arai Eri , Tian Ying , Gotoh Masahiro , Komiyama Motokiyo , Fujimoto Hiroyuki , Sakamoto Michiie , Kanai Yae 
Published: August 2015

Abstract
Background: We have identified CpG island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (ccRCCs) characterized by accumulation of DNA hypermethylation of CpG islands, tumor aggressiveness and poorer patient outcome. Furthermore, the criteria for prognostication of patients with ccRCCs have been established based on DNA methylation levels of ccRCC-specific CIMP marker genes. Recently, we have developed an anion exchange High Performance Liquid Chromatography (HPLC) column for detection of methylated DNA. To make the prognostication of patients with ccRCCs clinically applicable, the newly developed HPLC column has been applied to DNA methylation analysis of CIMP marker genes.
Methods: Genomic DNA extracted from tumorous tissue samples of nephrectomy specimens of 5 patients with CIMP-positive ccRCCs and 5 patients with CIMP-negative ccRCCs was amplified by Polymerase Chain Reaction (PCR) after sodium bisulfite conversion with the EpiTect Bisulfite Kit (QIAGEN). Three-hundred and eighty-four bp-PCR products encompassing the promoter region of the FAM150A gene, a CIMP marker gene, and including 39 CpG sites were subjected to the HPLC column. DNA methylation levels were quantitatively evaluated using 0% and 100% methylated DNA controls (QIAGEN).
Results: The analysis time for all samples was within 10 minutes. The retention time difference between 0% and 100% methylated DNA controls was 0.34 min. CIMP-negative ccRCCs were detected as a distinct single peak which had similar retention time with the 0% methylated DNA control, whereas CIMP-positive ccRCCs showed a bimodal peak pattern. Retention time of the later peak of CIMP-positive ccRCCs was similar with the 0% methylated DNA control. On the other hand, an early peak was eluted at various positions depending on its DNA methylation level. Based on the differences of chromatogram patterns, CIMP-positive ccRCCs were completely discriminated from CIMP-negative ccRCCs. DNA methylation levels of the FAM150A gene of each tissue sample calculated from retention time were consistent with those previously evaluated using MassARRAY system (Sequenom).
Conclusion: Using newly developed anion exchange HPLC column, tissue specimens of CIMP-positive ccRCCs were readily, quickly and clearly distinguished from those of CIMP-negative ccRCCs, indicating that this HPLC method may be suitable for clinical application of prognostication of patients with ccRCCs based on CIMP diagnosis.
Citation Format: Takuya Yotani, Yuriko Yamada, Eri Arai, Ying Tian, Masahiro Gotoh, Motokiyo Komiyama, Hiroyuki Fujimoto, Michiie Sakamoto, Yae Kanai. Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes using anion exchange high performance liquid chromatography. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1049. doi:10.1158/1538-7445.AM2015-1049