Isolation by size of epithelial tumor cells - A new method for the immunomorphological and molecular characterization of circulating tumor cells

We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, us ing peripheral blood sample volumes as small as 1 ml. Using this assay, epi thelial tumor cells can be isolated individually by filtration because of t heir larger size when compared to peripheral blood leukocytes. ISET paramet ers were defined using peripheral blood spiked with tumor cell lines (HepG2 , Hep3B, MCF-7, HeLa, and LNCaP), ISET can detect a single, micropipetted t umor cell, added to 1 mi of blood, We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor ce lls collected using ISET, Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate h omozygous p53 deletion in single Hep3B cells after filtration and laser mic rodissection. Finally, we provide evidence for the in vivo feasibility of I SET in patients with hepatocellular carcinoma undergoing tumor resection, I SET, but not reverse transcriptase-polymerase chain reaction, allowed analy sis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET con stitutes a novel approach that should open new perspectives in molecular me dicine.