Higher-Frequency Analysis of Low-Frequency Data

Abstract

Background: Cell-free serum DNA levels are increased in patients with cancer, and at least partially, these DNA fragments are derived from cancer cells. A few reports indicated that methylated serum DNA in patients with bladder cancer (BCA) is a useful non-invasive biomarker. The purpose of this prospective multicenter study was to validate earlier studies. Materials and Methods: In total, 227 consecutive participants (non-muscle invasive BCA, n=75; muscle-invasive BCA, n=20; transurethral bladder resection (TURB) without BCA, n=48; benign disease, n=31; healthy individuals, n=53), were recruited for this study. Cell-free serum DNA was isolated and digested with methylation-sensitive restriction-enzymes (Bsh1236I, HpaII and HinP1I) to quantify the amount of methylated (TIMP3, APC, RARB, TIG1, GSTP1, p14, p16, PTGS2 and RASSF1A) DNA fragments. Results: The amount of methylated DNA was usually small (<10%), and the methylation frequencies varied for different genes (e.g. frequent: TIMP3; moderate: APC, RARB, TIG1; infrequent: p16, PTGS2, p14, RASSF1A, GSTP1). Methylation levels at each gene site and the number of methylated genes were increased in BCA compared to healthy individuals, but were similar in BCA and patients with non-malignant disease. The number of methylated genes allowed for discrimination (62% sensitivity, 89% specificity) of BCA patients from healthy individuals. DNA hypermethylation was not correlated with advanced stage or grade in patients with BCA. Conclusion: The detection of hypermethylated DNA in serum allows for discrimination of patients with BCA and healthy individuals, but there is no difference between patients with BCA and those with non-malignant disease, thereby limiting its value as a non-invasive biomarker.