Abstract P2-02-01: Accurate identification of metastatic breast cancer using methylated gene markers in circulating free DNA in peripheral blood
Authors: Sukumar S , Fackler MJ , Lopez-Bujanda Z , Teo WW , Jeter S , Umbricht C , Visvanathan K , Wolff AC 
Published: August 2014

Abstract
Background: Preliminary studies from our lab have shown that a panel of methylation markers in tissue identifies 100% of tested breast cancer and 95% of tested DCIS, and has high accuracy in cells from ductal fluid and spontaneous nipple discharge1,2. Other groups have reported on the use of a single marker or a panel of markers to detect breast cancer in serum or plasma. Cell-free DNA studies in the peripheral blood of breast cancer patients with advanced disease or with early-stage disease after completion of local therapy support the hypothesis that methylated DNA markers in serum or plasma could be used to monitor response to therapy and for long-term surveillance. Validation studies to test these hypotheses have been hampered by assay methodological issues such as the very small amount of DNA shed in the serum by tumor compared to the total DNA shed by normal cells.
Methods: To overcome this problem, we developed a modified quantitative methylation-specific PCR that directly measures the number of copies of methylated DNA markers in a small aliquot of serum (Serum-QM-MSP) and robustly detects less than 25 copies of DNA in 300 µL of serum. We then conducted a genome-wide methylome analysis to identify key markers that are preferentially methylated in serum from women with breast cancer and compared the profiles to those from women with no breast cancer. We then analyzed 300 µL each of sera from 55 normal women (single time point) and 43 women with metastatic breast cancer using this newly developed panel of markers and the Serum-QM-MSP assay. We also examined changes after therapy in a subset of patients with metastatic disease.
Results: Methylation markers were quantitatively detected in sera of 39 out of 43 (91% sensitive) metastatic breast cancer patients with varying tumor burdens, and not in sera of any of 55 women (100% specific) for an AUC=0.95, using a laboratory threshold of 7.2 cumulative methylation units. 28 of the 43 patients had sampling repeated 3–5 weeks after therapy started. Sera from patients whose tumors regressed and from those that had stable disease showed a quantitative reduction, while those with progressive disease showed an increase in methylation levels of several genes.
Conclusion: Our results suggest that methylated DNA in serum accurately discriminates between blood samples from normal women and from metastatic breast cancer patients. Also, early changes after therapy initiation for metastatic disease may correlate with subsequent clinical outcome. Assay analytical validation studies are ongoing. Studies examining a potential role in surveillance in the adjuvant setting and therapeutic benefit in the metastatic setting are warranted.
1. Fackler MJ et al. Genome-wide methylation analysis identifies genes specific to breast cancer hormone receptor status and risk of recurrence. Cancer Res. 2011 Oct 1;71(19):6195–207. PMCID: PMC3308629. 2. Fackler MJ, et al. Hypermethylated genes as biomarkers of cancer in women with pathologic nipple discharge. Clin Cancer Res. 2009 Jun 1;15(11):3802–11. PMID: 19470737
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-02-01.