P4-07-06: Correlation of Two Analytical Methods for Circulating Tumor Cells in Peripheral Blood of Patients with Primary Breast Cancer.
Authors: Jaeger BAS , Rack B , Jueckstock J , Salmen J , Ortmann U , Lorenz R , Rezai M , Beck T , Schneeweiss A , Zwingers T , Beckmann MW , Friese K , Janni W 
Published: June 2012

Abstract
Background
While the evidence for circulating tumor cells (CTCs) as a prognostic marker in metastatic breast cancer has been well established, there is still a lack of data in primary disease. In the SUCCESS A trial two different techniques for the detection of CTCs in early breast cancer were prospectively evaluated.
Material and Methods: SUCCESS A compared FEC-Docetaxel vs. FEC-Docetaxel-Gemcitabine and 5 vs. 2 years of treatment with zoledronic acid in primary breast cancer patients and node positive or high-risk node negative disease. Two different techniques to detect CTCs were prospectively evaluated in two consecutive, but comparable subgroups of the whole study population.
In 3515 samples the CellSearch® System (Veridex, Warren, USA) was used for CTC detection. Immunomagnetic enrichment with an EPCAM-antibody was followed by labeling with monoclonal antibodies specific for cytokeratin (8, 18, 19) and leukocytes (CD45). 2165 samples were evaluated with a manual immunocytochemistry (MICC) protocol. Cytospins were prepared after mononuclear cell enrichment based on Oncoquick® centrifugation (greiner bio-one, Frickenhausen, Germany). Staining was performed with the monoclonal pancytokeratin antibody A45-B/B3 (Micromet, Munich, Germany) and the APAAP technique. Conventional light field microscopy (Axiophot; Zeiss, Oberkochen, Germany) was used for the detection of stained cells.
For both methods, the cut-off value for positivity was ≥ 1 CTC. All events were evaluated by two independent observers.
Results: CTCs were examined in a total number of 3243 patients before and after chemotherapy (CHT). The two subgroups evaluated with one or the other method were well-balanced regarding clinical parameters as tumor size, grading, lymph node-status, hormone receptors and Her2. Furthermore there was no significant correlation between the CTC positivity and one of these clinical parameters using CellSearch or the MICC, respectively (p &gt; 0,05 using the chi square test each time).
Before adjuvant CHT 21. 3% (424 out of 1994) and 21.1 % (264 out of 1249) of the patients were found positive for CTCs using CellSearch® or the MICC respectively, with a mean CTC level of 5.9 (range: 1 to 827) and 3.1 (range: 1 to 256).
Immediately after CHT 21.9% (333 out of 1521) and 16.5% (151 out of 916) of the patients were positive for CTCs using CellSearch® or the MICC. The mean CTC level decreased to 3.0 (range: 1 to 124) and 2.1 (range: 1 to 23) in both analytical methods.
Using CellSearch® there was a significant correlation between the presence of CTCs before CHT and disease progression (p = 0.0044), as well as survival (p = 0.0001), whereas the MICC did not predict any of these (p = 0.3143 and p = 0.0801 respectively; the chi-square test was used each time).
Conclusion: We found comparable prevalence of CTCs before and after adjuvant chemotherapy both with the CellSearch® System or the MICC. However, prognostic relevance could only be shown for CTCs detected with the CellSearch® System. This may be attributed to the high standardization and reproducibility of the automated system, as well as the additional CD45 counterstaining. According to our findings, the FDA approved CellSearch® System should be used as gold standard for CTC detection in future clinical trials.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-06.