1. Circulating tumor cells (CTCs) in blood of breast cancer patients: Cytological detection and technical characterization Enrica Bresaola, Mara Jo Miller, Marco Picozzi, Maria Teresa Sandri, Chiara Casadio ______________________ Cytology Unit, Department of Pathology & Laboratory Medicine European Institute of Oncology, Milan, Italy

2. Introduction  Circulating tumor cells (CTCs) were described for the first time more than one century ago and their occurrence in the bloodstream fit with the hematogenous theory of metastatization  Currently, their detection may play a pivotal role in the prognosis and prediction of therapy efficacy, providing us with insights into the clinical outcome, cell dissemination, drug resistance and treatment- induced cell death

3. Purpose  To assess the feasibility of detecting CTCs in blood samples of breast cancer patients using the Thin Prep® cytological preparation after a concurrent analysis with the CellSearch System (Veridex LLC, Warren, NJ)  To further characterize these cells according to estrogen receptor immunoreactivity and Her-2/neu gene status evaluation by fluorescence in situ hybridization (FISH) analysis

4. Materials and Methods  7.5 ml of whole blood were drawn from breast cancer patients into the CellSave Preservative Tube containing a cellular preservative and processed within 72 hours  The CellSearch Profile kit was then utilized to separate the CTCs by treatment with iron particles coated with antibodies against the Epithelial Cell Adhesion Molecule (EpCAM) for capturing CTCs  CTCs were then magnetically separated out and concentrated into a remaining aliquot of 1ml

5. Materials and Methods  This aliquot was centrifuged at 1700 rpm for 7 minutes and the supernatant discarded  The pellet was then added directly into the Preservcyt® vial for subsequent processing of ThinPrep slides  The slides were colored with H&E and evaluated microscopically  Estrogen receptor immunostaining and FISH analysis were carried out according to previously refined laboratory methods

6. Results  A total of 106 blood samples, where the CellSearch System obtained CTCs, were further evaluated cytologically  Of these, 60 were negative and 46 were positive for malignant cells (range: 1-615) (Figure 1)

7. Figure 1

8. Results  Immunocytochemistry for estrogen receptor was performed in 7 samples and only one case showed ER- positive tumor cells  10 cases were analyzed by FISH: 7 cases had no amplification while in 3 cases no more cells were detected +ve cell -ve cell

9. Conclusions  Cytological detection of CTCs in blood specimens from breast cancer patients can be useful in providing samples for testing predictive indicators of prognosis and clinical response during therapy