Evaluation of c-FLIP(long), c-FLIP(short), and caspase 8 expression in primary tumors using a semiquantitative multiplex LSC-based assay

Abstract

Abstract Recent studies have demonstrated the value in quantification of tumor expression and mutational status of growth factor signaling components as potential predictors of clinical response. Quantification of elements in the apoptosis pathways may provide a similar value in predicting clinical response to new therapies based on induction of apoptosis through death-receptor-mediated signaling. To that end, we have qualified a 5-color multiplexed immune histofluorescent tissue-based assay to provide semiquantitative measurement of expression of death-inducing signaling complex (DISC) components. The DISC components we measured in FFPE archival samples from human solid tumors are caspase 8, c-FLIP(long) and c-FLIP(short). Additional components of the multiplex were a pan-keratin reagent for identification of cytokeratin expressing epithelial tumor cells, and a histone H3 reagent for staining quality control. Image collection was accomplished via laser scanning cytometry (LSC) with a data pipeline which generates FCS3.0 (flow cytometry standard 3.0) files. This allowed intensity analysis of gated tumor enriched regions via standard flow cytometry analysis software. Antibodies were identified via in vitro screens using high/low target expressing cell lines, and further tested using xenografts of the cell lines of interest. The assay was qualified by examining day-to-day, slide-to-slide, and inter-operator variability using xenograft TMA (tissue micro-array) controls. The intensity of stained samples over 24 hours was stable. Total experimental %CV was demonstrated to be less than 25%. We analyzed 6 commercially available cancer TMA slides containing 387 tumor cores representing colon, lung, pancreas, breast, sarcoma, and non-Hodgkin's lymphoma, along with 43 corresponding normal cores. We found a wide range of expression of caspase 8, c-FLIP(l), and c-FLIP(s) within each tumor type, as well as clear differences between normal and tumor tissues. These findings indicate a potential for stratification of clinical response by DISC protein expression in therapeutics that act via death receptor mediated apoptosis.