Comparison of the Cellsearch Profile Kit (CPK) with the Standard Cellsearch Epithelial Kit (CEK) Demonstrates the CPK Produces Higher Circulating Tumor Cell (CTC) Yields and Is Better Suited for Use in Obtaining CTC's for Molecular Characterization.
Authors: Flores L. , Kindelberger D. , Ligon A. , Cappeletti M. , Fiorentino M. , Loda M. , Cibas E. , Janne P. , Krop I. 
Published: December 2010

Background: CTC offer a potential non-invasive approach to obtain and characterize metastatic tumor cells from solid cancers. However, conventional methods for isolating CTC, which use Epcam specific immunomagnetic beads, have been limited by low CTC yields and contaminating leukocytes. We sought to optimize and characterize CTC isolation using the CPK(Veridex LLC) and compare it to conventional methods using the CEK and automated CellTracks counting system, the FDA approved standard. We hypothesized that the CPK, which avoids the potential CTC loss and destruction associated with the multiple washes and labeling steps included in the CEK, would offer improved cell recovery.Methods: We collected two peripheral blood samples from patients (Pts) with metastatic breast cancer (n = 75) and processed them in parallel. Following automated enumeration of the samples with the CEK method, the cells were washed, fixed, and processed as cytospin slides followed by H&E and FISH. Uncounted samples processed with the CPK method were similarly washed, fixed, and processed as cytospin slides, followed by either H&E, IHC (AE1/AE3), IF (HER2, pHER2, EGFR, pEGFR) or FISH using probes against CEP7, EGFR, HER2, and MET.Results: The median cell count from pts with breast cancer using the CPK method was 120 per 7.5 mL of blood (range 4-4967) compared to 4 (range 0-57) using the CEK method (p &lt; 0.0001; t-test). The purity of these cells was high: 59.7 % of cells isolated by CPK (range: 20.9-95.0) were identified as tumor cells by cytokeratin staining while 6.4% (range: 0-21.8) were identified as WBCs by CD45 staining. The remaining 32.3% (range: 4.8-68.0) of cells stained with DAPI only and could not be classified as either tumor cells or lymphocytes, and appeared to have lost their membrane during the isolation. We confirmed the purity of the CTC by HER2 FISH. Among pts whose primary cancer was HER2+ (n=70), 72.4% (range: 44-100%) of CTC had an HER2 gene copy number &gt; 4. From samples isolated from healthy controls (n = 40) and processed using the CPK method the mean total number of cells was 5 per 7.5 mL of blood (range 0-20). These cells were positive for CD45 with only 2 copies of HER2 by FISH. The CPK had low intrapatient variability (CV 9.6%) and samples were stable for at least 72hr. Recovered CTC were well suited for evaluation by IF for phospho antibodies (e.g. pHER2, pAkt), TUNEL (apoptosis), and Ki67 (proliferation). Studies using BC cell lines demonstrated that viable cells could be recovered and propagated with an input as few as 50 cells consistently yielding viable cultures.Conclusion: The CPK technique's relatively high yield, purity, and sample stability make it well suited for use in obtaining CTC's for molecular characterization, including FISH and IF. Assays for mutation detection are currently being evaluated. This technology potentially has a large number of applications in investigating the biology of metastatic cancer as well as in drug development where it can be used to identify predictive biomarkers, mechanisms of resistance, and facilitate pharmacodynamic studies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3003.