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The aim of this study was to develop a new methodology that is suitable for  DNA  methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion‐exchange column for high‐performance liquid chromatography ( HPLC ) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The  DNA  methylation levels of synthetic  DNA  were quantified accurately and reproducibly within 10 minutes without time‐consuming pretreatment of  PCR  products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic  DNA  fragments. When the  DNA  methylation status of the   FAM 150A  gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (cc RCC ), was examined in 98 patients with cc RCC , bulk specimens of tumorous tissue including cancer cells showing  DNA  methylation of the   FAM 150A  gene were easily identifiable by simply viewing the differentiated chromatograms, even when the cancer cell content was low. Sixteen cc RCC  showing  DNA  methylation more frequently exhibited clinicopathological parameters reflecting tumor aggressiveness (ie, a larger diameter, higher histological grade, vascular involvement, renal vein tumor thrombi, infiltrating growth, tumor necrosis, renal pelvis invasion and higher pathological  TNM  stage), and had significantly lower recurrence‐free and overall survival rates. These data indicate that  HPLC  analysis using this newly developed anion‐exchange column could be a powerful tool for  DNA  methylation diagnostics, including prognostication of patients with cancers, in a clinical setting.

Novel method for DNA methylation analysis using high‐performance liquid chromatography and its clinical application