Supplementary Material for: Methylated DNA and Total DNA in Serum Detected by One-Step Methylation-Specific PCR Is Predictive of Poor Prognosis for Breast Cancer Patients

<b><i>Purpose:</i></b> We recently developed the one-step methylation-specific PCR (OS-MSP) assay which can detect methylated DNA (met-DNA) in serum with high sensitivity. To examine its prognostic value, we applied this new assay to the detection of met-DNA in serum of breast cancer patients. <b><i>Methods:</i></b> Serum samples taken before surgery from 336 primary invasive breast cancer patients were subjected to the OS-MSP assay for the promoter regions of <i>GSTP1</i>,<i> RASSF1A</i>, and <i>RAR</i>β<i>2</i>. The assay outcome was considered positive when methylation was detected in at least one of these three genes. Total DNA content in serum was also determined. <b><i>Results:</i></b> Of the 336 stage I/II patients, 33 (10%) were positive for met-DNA in serum and showed a significantly worse overall survival (OS) rate at 100 months (78 vs. 95%; p = 0.002) than those with negative findings (n = 303). Patients with high total DNA in serum (n = 112) also showed a significantly worse OS rate at 100 months (86 vs. 97%; p = 0.001) than those with low total DNA in serum (n = 224). Moreover, patients both positive for met-DNA and with high total DNA in serum (n = 18) showed a much worse OS rate at 100 months (65 vs. 94%; p &lt; 0.001) than the others (n = 318). <b><i>Conclusions:</i></b> Met-DNA in serum detected with the OS-MSP assay constitutes a significant and independent prognostic factor, and its combination with total DNA in serum seems to be even more effective for prediction of prognosis for breast cancer patients.