Promoter hypermethylation, a widely studied epigenetic event known to influence gene expression levels, has been proposed as a potential biomarker in multiple types of cancer. Clinical diagnostic biomarkers are needed for reliable prediction of bladder cancer recurrence. In this paper, DNA promoter methylation of five C-terminal Ras-association family members (RASSF1A, RASSF2A, RASSF4, RASSF5, and RASSF6) was studied in 64 formalin-fixed paraffin-embedded (FFPE) bladder cancer and normal adjacent tissues using methylation-specific high-resolution melting (MS-HRM) analysis. Results showed that 73% (30/41) of transitional cell carcinoma, 100% (3/3) of squamous cell carcinoma, and 100% (4/4) of small cell carcinoma demonstrated promoter methylation of the RASSF1A or RASSF2A gene, but only 6% (1/16) of normal tissues had promoter methylation of RASSF genes. Testing positive for hypermethylation of RASSF1A or RASSF2A promoter provided 77% sensitivity and 94% specificity for identification of cancer tissues with an area under the curve of 0.854, suggesting that promoter methylation analysis of RASSF1A and RASSF2A genes has potential for use as a recurrence biomarker for bladder cancer patients. 1. Introduction In 2011, about 52,000 men and 17,000 women will be diagnosed with bladder cancer in the United States. Before a normal cell transforms into a bladder cancer cell, a series of molecular alterations are accumulated to initiate the process of transformation. Although we do not fully understand the mechanisms, DNA alterations including hypermethylation and somatic mutation are commonly observed events in human cancer. In a recent bladder cancer study FGFR3 mutation in combination with APC, RASSF1A, and SFRP2 methylation markers provided a sensitivity of 90% using tissue samples and 62% using paired urine samples to identify the presence of cancer with 100% specificity [1]. In nonsmall cell lung cancer (NSCLC) and breast cancer, studies showed that RASSF1A had different frequencies of methylation depending on histology [2, 3]. In nasopharyngeal carcinoma, RASSF2A was frequently inactivated by its promoter methylation and the methylation correlated with lymph node metastasis [4]. The Ras-association family, also called RASSF tumor suppressor genes, currently includes 10 members. All of the RASSF proteins contain a Ras-association domain on their C-terminus (RASSF 1–6) or N-terminus (RASSF 7–10). Two important issues that are not previously addressed by studies of RASSF gene methylation are (1) whether all of the RASSF family members show aberrant methylation
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