Association of serum tumor-related methylated DNA with circulating tumor cells in peripheral blood of breast cancer patients.
Authors: Van der Auwera I , Elst H , Van Laere S , van Dam P , Van Marck E , Vermeulen P , Dirix L 
Published: December 2010

Abstract
Abstract #5017
Circulating tumor cells (CTC) and free tumor-related methylated DNA in blood have been separately associated with poorer disease outcome in breast cancer patients. However, no studies have looked into the relation between both molecular markers in breast cancer. In this study, we investigated the correlations between plasma total DNA, serum methylated DNA and CTC in blood from breast cancer patients.&#x2028; We simultaneously obtained matched triplets of peripheral blood, plasma and serum samples from 4 patients with localized breast cancer (group A), 59 patients with metastatic disease under treatment (group B), 16 untreated patients with metastatic disease (group C) and 20 healthy controls. CTC levels in blood were measured with the CellSearch System (Veridex). Plasma total DNA levels were determined by a qPCR method. Sera were analyzed by methylation-specific qPCR for three methylated markers: APC, RASSF1A and ESR1.&#x2028; Plasma total DNA levels in breast cancer patients were significantly increased when compared to healthy controls (P&lt;0.001). Differences between total DNA levels in different patient groups also reached statistical significance (P=0.04). The largest differences were measurable between groups A and C (median of 7 vs 29 ng/ml) (P=0.02) and groups B and C (median of 12 vs 29 ng/ml) (P=0.06). The sensitivity and the specificity of the total DNA assay for predicting malignancy was respectively 72.5% and 85%. Total DNA levels correlated with CTC (r=0.418, P&lt;0.001) and patient age (r=0.298, P=0.01), but not with other clinical variables. Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in 3 (16%) serum samples of healthy controls (P=0.003). APC was hypermethylated in 23 (29%), RASSF1A in 28 (35%) and ESR1 in 16 (20%) breast cancer cases. CTC were detected in 19 (70%) patients with serum methylated DNA and in 8 (30%) patients without methylated DNA (P=0.03). The presence of methylated DNA markers was only associated with ER status. On univariate analysis, the detection of CTC, high levels of circulating DNA, methylation of RASSF1A and the combinations of APC or RASSF1A methylation with ESR1 methylation were significantly associated with progressive disease. Multivariate analysis indicated that only RASSF1A methylation, high total DNA levels, HER2 and PR status were significantly associated with disease status.&#x2028; The number of CTC was significantly increased in patients with high levels of total DNA and in patients with at least one methylated DNA marker in serum, suggesting that CTC might be a potential source of circulating tumor-related DNA. Only RASSF1A methylation and high DNA levels were independently associated with progressive disease. The combination assay of CTC and serum methylated DNA did not enable a more informative assessment of disease status.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5017.