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

Our plain-English summary of Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions, by H.P. Dong, A.K. Ree Rosnes, A.J. Bock, et al (Cytopathology, 2011).

The study

• Authors: H.P. Dong, A.K. Ree Rosnes, A.J. Bock, A. Holth, V.A. Flørenes, C.G. Trope', B. Risberg, B. Davidson
• Journal: Cytopathology
• Published: 2011;22(6):373-82
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

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.

In plain English

This laboratory study from the Norwegian Radium Hospital in Oslo developed a flow cytometry test to measure c-FLIP in fluid collected from body cavities (effusions). c-FLIP is a protein that can block one pathway of programmed cell death. The study did not examine asbestos exposure. A malignant mesothelioma cell line (MSTO 211H) was one of five cell lines used to set up the test. After comparing two antibodies and three cell-preparation methods, the authors chose an Abcam antibody and the IntraStain kit. They then tested 69 effusions (61 peritoneal, 8 pleural) from 65 patients with ovarian carcinoma, collected in 2002 to 2006. c-FLIP was found in tumour cells in all 69 effusions, in 21 to 100% of cells (median 80%). It was not related to effusion site, age, grade, stage, previous chemotherapy or response to chemotherapy. It was also not related to overall survival (P = 0.873) or progression-free survival (P = 0.513). In a limited analysis of 21 matched cases, c-FLIP was inversely related to cleaved caspase-3 (P = 0.029). The authors concluded that c-FLIP is frequently expressed in these effusions but appears unrelated to disease aggressiveness.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory method-development study (antibody testing, titration and comparison of fixation and permeabilization protocols in cell lines), followed by analysis of clinical effusion specimens for association with clinical features and survival
• Who was studied: 69 malignant effusions (61 peritoneal, 8 pleural) from 65 patients diagnosed with ovarian carcinoma, submitted to the Norwegian Radium Hospital, Oslo, in 2002 to 2006. The test was first developed on five cell lines: breast carcinoma (T47-D), ovarian carcinoma (OVCAR-3, OVCAR-8, SKOV-3) and mesothelioma (MSTO 211H).
• Main results:
  • Of the antibody and preparation methods tested, the authors chose the IntraStain kit for the clinical specimens. The Santa Cruz antibody was excluded because it did not clearly detect the expected bands.
  • c-FLIP was detected in tumour cells in all 69 ovarian carcinoma effusions, in 21 to 100% of tumour cells (median 80%).
  • c-FLIP expression was not related to effusion site, patient age, histological grade, FIGO stage, previous chemotherapy, or response to chemotherapy at diagnosis and at first recurrence (P greater than 0.05).
  • Among the 65 patients, followed for 1 to 156 months, c-FLIP expression was not related to overall survival (P = 0.873) or progression-free survival (P = 0.513).
  • Results were similar when the survival analysis was limited to 47 patients with effusions taken at primary diagnosis before chemotherapy (P = 0.605 for overall survival and P = 0.872 for progression-free survival).
  • In a limited analysis of 21 matched cases, the authors compared c-FLIP with the cleaved caspases and dUTP incorporation measured earlier, and observed an inverse relationship between c-FLIP and cleaved caspase-3.
  • The inverse correlation between c-FLIP and cleaved caspase-3 had P = 0.029 (paired sample t-test). There was no association with the other previously studied apoptosis markers. Across all these marker comparisons, 21 to 28 effusions were matched.
  • No association was observed between c-FLIP and death receptor expression, based on 28 effusions analysed in both studies.
• The authors' conclusion: The authors concluded that they had established an assay for measuring c-FLIP in cytology specimens. They found that c-FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.
• Limitations the authors state:
  • The lack of association between c-FLIP and death receptor expression was based on a small series of 28 effusions.
  • The cleaved caspase analysis was done earlier than the c-FLIP analysis, and the difference in storage time of the frozen samples may affect results.
  • The inverse association with cleaved caspase-3 and the lack of association with death receptors need to be tested in a larger case series.
  • The assay may need to be calibrated for analysis of different types of clinical specimens.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Ovarian cancer, Pleural plaques and thickening.

Medical terms in this study, explained in our Lexicon: Carcinoma, Apoptosis, Malignant Mesothelioma, Cytology.

Related studies

• Death receptor 5 and cellular FLICE-inhibitory protein regulate pemetrexed-induced apoptosis in human lung cancer cells — L. Su, G. Liu, et al. (2011)
• PINCH-2 expression in cancers involving serosal effusions using quantitative PCR — Y. Yuan, H.P. Dong, et al. (2011)
• Flow cytometric analysis of ploidy in immunohistochemically confirmed examples of malignant epithelial mesothelioma — H.F. Frierson, S.E. Mills, et al. (1988)
• Expression of the Ets transcription factor EHF in serous ovarian carcinoma effusions is a marker of poor survival — K. Brenne, D.A. Nymoen, et al. (2012)
• Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent — J.L. Hurwitz, I. Stasik, et al. (2012)
• Potentiation of cisplatin and carboplatin cytotoxicity by amphotericin B in different human ovarian carcinoma and malignant peritoneal mesothelioma cells — L. Poulain, F. Sichel, et al. (1997)

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