Abstract 841: Comparison of two molecular assays for the detection of circulating tumor cells in blood of primary breast cancer patients and their correlation with disseminated tumor cells in the bone marrow
Authors: Strati Areti , Markou Athina , Hoffmann Oliver , Kimmig Rainer , Lianidou Evi S. , Kasimir-Bauer Sabine 
Published: February 2011

Abstract
Background: In primary breast cancer, the presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor, while the role of circulating tumor cells (CTCs) in blood still has to be proven since a standard method for their detection has not been defined. Molecular approaches are generally more sensitive than cytometric methods and have the additional advantage of a high-throughput technology. Therefore, we examined the presence of CTCs in blood by using two different molecular assays and assessed their correlation with the detection of DTCs in BM of primary breast cancer patients.
Patients and Methods: In total, 418 blood samples and 384 BM samples of 377 primary breast cancer patients were studied. Two x 5 ml blood samples were collected and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads. All samples (same cDNAS) were further analyzed by using: a) the AdnaTest BreastCancer (AdnaGen AG) for the detection of three breast cancer associated markers: GA733-2, MUC-1, HER-2 and beta Actin (internal reference gene control), analyzed by capillary electrophoresis on the Agilent Bioanalyzer 2100 and b) real time quantitative RT-PCR for CK-19 mRNA in the Light-Cycler System (Roche Diagnostics). Furthermore, 384 bilateral BM aspirates were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 and DTCs were evaluated with the ARIOL system (Applied Imaging) according to the ISHAGE evaluation criteria.
Results: DTCs were found in 115/384 BM samples, resulting in a positivity rate for DTCs of 30%. Applying the AdnaTest, 409/418 samples could be evaluated for the presence of CTCs. 53/409 (13%) samples had detectable CTCs expressing MUC-1 (30%), GA733-2 (25%) and HER2 (70%), respectively. CK19 RT-PCR could be performed in 411/418 samples with a positivity rate of 21% (86/411 samples). A comparison of both molecular CTC assays could be performed in 400 samples with a concordance rate of 71% resulting in 274 blood samples found to be negative by both assays, 11 samples found to be positive by both assays, AdnaTest+/CK19- was documented in 42 cases and AdnaTest-/CK19+ in 73 cases, respectively. Concordant results between the detection of DTCs in the BM and CTCs were found in 65% (AdnaTest) and 62% (CK19 RT-PCR) of all cases, respectively.
Discussion: (1) Molecular CTC assays gave different results in 115/400 cases (28.7%) in the same cDNA samples. This could be attributed to the fact that the evaluation of CTCs positivity between these tests is based on a totally different set of genes. (2) Our results might indicate the heterogeneity of CTCs in breast cancer. (3) The low correlation between the detection of DTCs and CTCs by both methods indicates that CTCs may provide prognostic information complementary to DTCs in BM.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 841.