Title:	A novel blood test for detection of viable tumor cells in blood of patients with epithelial cancers.
Authors:	Wen-Tien Chen(1,2) & Yunyun Yeh(2) (1) Department of Medicine, Stony Brook University, State University of New York, Stony Brook NY 11794; (2) Vitatex Inc., 25 Health Sciences Drive, Stony Brook, NY 11790.
Presented At:	Abstract for the third EDRN-Gordon Research Conference (GRC) on New Frontiers in Cancer Detection and Diagnosis in Santa Barbara, CA.
Date Presented:	January 16-21, 2005
Abstract
INTRODUCTION: It has become increasingly clear that the metastatic potential of a solid tumor correlates best with the presence of these cells in the systemic circulation. However, molecular analyses of these cells remains technically challenging because these emigrating tumor cells are few in number relative to the enormous numbers of normal blood cells and because many of the tumor cells found in peripheral blood are nonviable. METHODS: We have developed a novel cell separation technology based on preferential adhesion and degradation of tumor cells to cell adhesion matrix (CAM) coated surfaces. Viable tumor cells attach to CAM with great avidity, but normal cells (including more than 99.9% of white cells and 99.9999% of red cells) and dead or dying tumor cells do not. The CAM technology can also be used to mark tumor cells for the purpose of cancer identification. When the CAM is prepared using fluorescently labeled collagen, the invasive tumor cells become labeled, since they exhibit a propensity to bind and ingest collagen. In contrast, normal cells leave the CAM undisturbed. Immunocytochemistry and real-time RT-PCR using multiple epithelial/tumor associated cell lineage markers were used to validate the CAM-recovered cells from blood as circulating tumor cells. CAM recovered cells were viable based on Molecular Probes LIVE/DEAD Viability testing. RESULTS: 10 mL of blood from 125 patients with metastatic cancer was collected and analyzed using the CAM technique. One million-fold enrichment of viable human tumor cells from the peripheral blood of healthy donors in model experiments, as well as in patients with various metastatic diseases can be achieved in a single step. Cells were identified with multiple criteria (positive for epithelial/tumor markers, and uptakes of CAM fragments and nucleic acid dyes) as viable circulating tumor cells. Our current assays successfully detected viable tumor cells in blood samples from patients with stage I to IV non-small-cell lung cancer, invasive breast ductal carcinoma, adenocarcinoma of the ovary, pancreas, colon, rectum, liver, and prostate. Tumor cells were found, by a microscopic cell counting method, in the range of 10 to 500 cells per mL of blood for positive samples. In contrast, none of any of the healthy donors and patients with inflammatory lung diseases yielded a (false) positive result. CONCLUSION: Cell separation based on preferential adhesion of tumor cells to CAM coated surfaces is a rapid and reliable method of detecting viable circulating tumor cells from blood in humans.