68-4

50/136

370CellSearch® system (MENARINI, Itary) has good reproducibility and can detect even a single CTC in a 7.5-ml peripheral blood sample7). In breast, colorectal, and prostate cancers, CTC counts before treatment and after the initial therapy correlate strongly with progression-free survival and overall survival (OS), moreover, therapeutic effects and prognosis can be predicted by measuring the CTCs count4-9). As a result, the CellSearch® system has been approved by the United States Food and Drug Administration (FDA) to test for CTCs in breast, prostate, and colorectal cancers. This study aimed to investigate whether detection of CTCs with the CellSearch® system could similarly be useful to predict prognosis in esophageal cancer.PatientsThis study involved 38 esophageal cancer patients treated at the Juntendo University Hospital Depart-ment of Esophageal and Gastroenterological Surgery from May 2010 to April 2013. All patients had been pathologically diagnosed with esophageal cancer before treatment. The exclusion criteria are as follows: (1)multiple primaries (multicentric esoph-ageal cancer are included); (2) non primary cases; (3) history of any cancer within 5years.Written informed consents were obtained from all enrolled patients before this study. This study was approved by the Ethical Committee of Juntendo University Hospital (No.12-80).Clinicopathological data were retrospectively retrieved from our database and electronic medical records. Tumor stage was assessed according to the International Union Against Cancer (UICC) TNM classification 7th edition for esophageal cancer10) from findings of gastrointestinal endos-copy, upper gastrointestinal series, computed tomography, and endoscopic ultrasound.Measurement of Circulating Tumor CellsA CellSearch® system epithelial cell kit was used to detect rare CTCs in whole blood by immuno-magnetic separation. The kit contains a magnetic bead-based capture reagent and an immunofluo-rescent staining reagent, and the magnetic beads contain nanoparticles with magnetic cores surrounded by a polymer layer coated with an epithelial cell adhesion molecule (EPCAM) anti-body to enrich CTCs. After enrichment and concen-tration using immunomagnetic separation, staining reagents are added to detect CTCs. Anti-CK-PE is specific for the intracellular protein cytokeratin (specific to epithelial cells), a nucleic acid stain (4′,6-diamidino-2-phenylindole [DAPI]) stains the cell nuclei, and anti-CD45 and APC react specifi-cally with leukocytes.The reagent and sample mixture are placed in a cartridge that sits in a MagNest® holder that gener-ates a magnetic field using the CellTracks Auto-Prep system (MENARINI, Itary). Magnetically labeled epithelial cells move to the surface of the cartridge due to the strong magnetic field gener-ated by the MagNest®, the fluorescent images are captured by the Cell Tracks® Analyzer II, and candidates stained with both CK-PE and DAPI inside the cartridge are displayed. Images are presented in a gallery format for the final cell clas-sification. The images are classified as tumor cells based on morphology and phenotype (EPCAM+, CK+, DAPI+, and CD45−).A 20-ml blood sample was collected before all treatment, and 10 ml was distributed among two Cell Save storage tubes. Subsequently, 7.5 ml of the 10 ml blood sample was transferred to a conical test tube, 6.5 ml of diluent was added, and the conical test tube was capped and mixed by inverting five times. The sample was centrifuged at 800 rpm for 10 min with the centrifuge brake released. The sample was placed in the CellTracks AutoPrep device of the CellSearch® system for processing within 1 hour.The CellTracks® AutoPrep system specifically isolates and extracts epithelial cells from the many cells in the blood using magnetic particles that consist of antibodies for EPCAM bound to iron nanoparticles. The isolated epithelial cells are bound by a fluorescently labeled cytokeratin mono-clonal antibody, and the nuclei are stained with DAPI fluorescent DNA stain. Similarly, leukocytes are bound by fluorescently labeled CD45 antibodies to distinguish them from CTCs. The CTC reaction solution is transferred into a cartridge that sits in a device with a fixed magnet called the MagNest and then placed in the CellTracks Analyzer II® of the CSS to analyze the test results. The magnetic force generated by the magnet in the MagNest moves the CTCs captured by the ferrofluid to the top of Methods