Abstract P3-02-14: Detection of Circulating Tumor Cells and HER2 Gene Amplification Status Using a Novel Microfluidic Platform (Cell Enrichment and Extraction Technology, CEE™)
Authors: Krishnamurthy S , Bischoff FZ , Mikolajczyk S , Lucci A. 
Published: April 2011

Abstract
INTRODUCTION: The rarity of circulating tumor cells (CTCs) in blood poses challenges in their detection. The majority of the techniques utilized for the detection of CTCs are limited in their ability to allow detailed phenotypic and genotypic evaluation of the CTCs. In this pilot study, we report the utility of a novel microfluidic platform utilizing cell enrichment and extraction technology (CEE™, Biocept, Inc., San Diego) for detection of CTCs in breast cancer and subsequent evaluation of HER2 gene amplification status of the CTCs by fluorescent in situ hybridization (FISH).
METHODS: Peripheral blood (10 mL) was collected from patients with operable breast cancer in a prospective institution review board approved protocol. Mononuclear cells were recovered using a Percoll density gradient method, incubated with a mixture of 10 primary capture antibodies (cAb) and then introduced into microchannels (CEE channels). The cells in the channels were stained using fluorescent pancytokeratin (CK) in addition to a fluorescent probe to detect cAbs on the cells. Cells were stained for CD45 as a negative control. The enumeration and localization of cAb-CK +, CD45- as well as cAb-CK-, CD45- cells was performed by microscopic examination of the microchannels. Finally the microchannels were processed for multicolor FISH using three direct labeled probes specific to centromere 8 (spectrum aqua), 17 (spectrum green) and HER2 (spectrum orange). The ratio of HER2:CEP17 &gt;2.2 in any CD45 negative cell was regarded as positive for HER2 gene amplification. RESULTS: Peripheral blood from 25 patients with T1N0 (12), T1N1 (3), T2N0 (3), T2N1 (2), T2N3 (1), T3N0 (1), T4N0 (2), T4N3 (1) showed cAb-CK+, CD45- CTCs in 75% (9/12), 66.6% (2/3), 33.3% (1/3) of the first three, respectively, and in all the patients in the remaining groups. Amongst the different genomic types of the primary tumor, cAb-CK+, CD45- CTCs were detected in 79% (15/19) Luminal A, 66% of Luminal B (2/3), 50% (1/2) of HER+ and 100% (1/1) of triple negative tumors. Overall 5/25 of the primary tumors were HER2 amplified. While cAb-CK+ cells were isolated in 3/5 of these patients, HER2 amplification was detected in a cAb-CK-, CD45- CTC in only one patient with a Luminal B tumor. In addition, HER2 amplified cAb-CK-CTCs were also detected in a single patient with primary tumor negative for HER2. The two patients with HER2+ CTCs were therefore cAb-CK-, CD45-. CONCLUSION:
1. The cell enrichment and extraction microfluidic technology (CEE™) provides a sensitive platform for enhanced detection and characterization of antibody-CK positive and negative CTCs.
2. This platform allows evaluation of HER2 gene amplification status by FISH in intact CTCs within the microchannels.
3. The utility of this platform for phenotypic and genotypic characterization of CTCs in breast cancer needs to be tested in larger clinical trials.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-14.