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.
© 2011 Blackwell Publishing Ltd.