408 Enrichment of tumor cells for cell kinetic analysis in human tumor biopsies using cytokeratin gating

Abstract

The feasibility of using cytokeratin antibodies to distinguish nonnal and malignant cells in human tumor biopsies using flow cytometry was tested for increasing the accuracy of cell kinetic measurements. Four different antibodies were screened on a 48 tumors from two centres; 26 head and neck (Amsterdam) and 22 oesophagus (Leuven). First screening was done by immunohistochemistry (ICC) on frozen sections to test staining intensity and fraction of cytokeratin-positive (CK+) tumor cells. The antibody showing the most positive staining was then used for flow cytometry: Two broad spectrum antibodies (AE1/AE3, E3/C4) showed the best results with ICC (>90% tumors positive). Cell suspensions for flow cytometry could be made from frozen material by a mechanical method. Enzymatic methods failed for frozen material but were best for fresh material. Tumor cell enrichment was tested by gating on CK+ cells in flow cytometry using the best antibody. Average enrichment was 0. 58 for head and neck tumors and 0.75 for oesophagus tumors; overall enrichment 0.65 (0 = no enrichment, 1 = pure tumor population). In several cases, 100% enrichment was achieved. We conclude that this method can significantly reduce errors in tumor cell kinetic measurements by reducing nonnal cell “contamination”.