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(American Journal of Pathology. 2000;156:57-63.)
© 2000 American Society for Investigative Pathology

Technical Advances

Isolation by Size of Epithelial Tumor Cells

A New Method for the Immunomorphological and MolecularCharacterization of Circulating Tumor Cells

Giovanna Vona^*, Abdelmajid Sabile^*, Malek Louha^*, Veronique Sitruk, Serge Romana^§, Karin Schütze^¶, Frédérique Capron^||, Dominique Franco^**, Mario Pazzagli, Michel Vekemans^§, Bernard Lacour, Christian Bréchot^* and Patrizia Paterlini-Bréchot^*

From INSERM U 370,^*
Faculté Necker, Paris, France; the Service de Gastroentérologie,
Hôpital Jean Verdier, Bondy, France; the Service de Cytogénétique,^§
Hôpital Necker, Paris, France; P.A.L.M. Mikrolaser Technologie,^¶
Munich, Germany; the Service d’Anatomie Pathologique^||
and the Service de Chirurgie Digestive,^**
Hôpital Antoine Béclère, Clamart, France; the Clinical Biochemistry Unit,
Department of Clinical Physiopathology, University of Florence, Florence, Italy; and the Service de Biochimie A,
Hôpital Necker, Paris, France

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, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

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