Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Flørenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE‐inhibitory protein (c‐FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c‐FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c‐FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c‐FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal‐to‐noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c‐FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c‐FLIP by Abcam and the IntraStain kit by Dako performed best. c‐FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c‐FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c‐FLIP levels and expression of the previously studied apoptosis marker cleaved caspase‐3 (P = 0.029). Conclusions: An assay for measuring c‐FLIP in cytology specimens is presented. c‐FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.