To improve the sensitivity of the panel, we combined DKK3 and ITIH5 with the potential biomarker RASSF1A. RASSF1A methylation was significantly different between serum of breast cancer patients (64 (47.1%) of 136), and both healthy controls (35 (25.9%) of 135) (P = 0.0035) and benign disease controls (six (15.4%) of 39; P = 0.0013). This evidence concerns the gene RASSF1 and breast cancer.