Determining the Recovery Efficiency and Relative Enrichment of Circulating Tumor Cells Using the CellSearch System in Patients with Metastatic Lung Cancer
DW Kindelberger, LM Flores, ES Cibas, AH Ligon, MF Loda, IE Krop, PA Janne. Brigham and Women's Hospital, Boston, MA; Dana-Farber Cancer Institute, Boston, MA
Background: Circulating tumor cells (CTCs) in the peripheral blood of patients with metastatic cancers have been increasingly accepted as indicators of prognosis and treatment response. Applications for purified CTCs, including fluorescence in situ hybridization (FISH) and direct sequencing, continue to increase. The CellSearch platform (Veridex, LLC), begins with a tube of peripheral blood and generates a sample enriched for CTCs. The CTCs can be either processed for counting or left uncounted for downstream applications. This study characterized both counted and uncounted samples, using H&E staining, immunohistochemistry (IHC), and FISH to evaluate the absolute number of CTCs and their relative enrichment.
Design: Peripheral blood samples were collected from 21 patients with biopsy-confirmed metastatic lung adenocarcinoma (three 7.5 ml tubes/patient). One tube was processed for counting and the remaining two tubes were processed uncounted for FISH and IHC, according to CellSearch protocols. Following enumeration of the counted samples with the CellTracks system, the cells were washed, fixed, and processed as cytospin slides followed by H&E staining. Uncounted samples were similarly washed, fixed, and processed as cytospin slides, followed by either H&E staining, IHC (using AE1/AE3), or FISH using probes against CEP7, EGFR, and MET. Cell numbers were determined by manually counting H&E-stained and FISH slides. CTCs were identified by AE1/AE3 IHC if they demonstrated circumferential membrane staining. CTCs were identified by FISH as those cells having greater than 2 signals with the CEP7 and/or EGFR/MET probes.
Results: For all cases, the number of tumor cells determined by H&E staining (mean 10.5) was significantly greater than the number determined by the CellTracks system enumeration (mean 1.8). Additionally, in all cases, the number of cells in counted samples was less than that seen in corresponding uncounted samples. The AE1/AE3 IHC and FISH results were concordant and showed that CTCs made up over 50% of the isolated cells.
Conclusions: Samples processed using the CellSearch system are significantly enriched for tumor cells. The CellTracks enumeration system appears to underestimate the true number of circulating tumor cells. Uncounted samples are sufficiently enriched for CTCs to allow for numerous downstream applications such as FISH and direct sequencing.
Tuesday, March 10, 2009 2:15 PM
Platform Session: Section G, Tuesday Afternoon