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

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

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

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

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

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)

Figure 1

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

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