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.

**研究目的**：本课题组近期研发了一步法甲基化特异性聚合酶链反应（one-step methylation-specific PCR, OS-MSP）检测方法，该方法可高灵敏度地检测血清中的甲基化脱氧核糖核酸（methylated DNA, met-DNA）。为评估该方法的预后价值，我们将这一新型检测方法应用于乳腺癌患者血清中的met-DNA检测。

**研究方法**：收集336例原发性浸润性乳腺癌患者的术前血清样本，针对GSTP1、RASSF1A及RARβ2基因的启动子区域开展OS-MSP检测。若在上述三个基因中至少一个检测到甲基化，则判定该检测结果为阳性。同时，对所有样本的血清总DNA含量进行测定。

**研究结果**：在336例Ⅰ/Ⅱ期患者中，33例（10%）血清met-DNA检测呈阳性，其100个月总生存期（overall survival, OS）率为78%，显著低于检测结果阴性者（303例，95%；p=0.002）。血清总DNA含量较高的患者（112例）100个月总生存期率为86%，同样显著低于总DNA含量较低的患者（224例，97%；p=0.001）。此外，同时满足血清met-DNA阳性且总DNA含量较高的患者（18例）100个月总生存期率仅为65%，远低于其余患者（318例，94%；p<0.001）。

**研究结论**：经OS-MSP方法检测得到的血清met-DNA是一项具有统计学意义的独立预后因素，将其与血清总DNA含量联合应用，或可更有效地预测乳腺癌患者的预后情况。