Discussion

Y Beiter, F Treindl, N Johanna, H Neubauer, C Sachse, MF Templin

Natural and Medical Sciences Institute (NMI) at the University of Tübingen; University of Tübingen; University of Düsseldorf; NMI TT Pharmaservices Berlin, Germany

The analysis of cellular signaling cascades is essential for understanding the processes that underlie drug response and drug resistance. Profiling of central signalling cascades requires the detection of protein expression and activation, which is difficult to achieve comprehensively. Our novel DigiWest technology combines the principles of Western blotting with a multiplexed Luminex bead array as a readout system. The system allows the generation of currently up to 600 of Western blot equivalents from a few micrograms of protein sample. Thereby, information on the expression and modification of hundreds of proteins is obtained with good reproducibility and linearity in combination with a large dynamic range.
Applying the DigiWest method, we analysed 24 fresh frozen tumor specimens from relapsed vs cured ovarian cancer patients directly on the protein level. A total of 466 antibodies per sample were employed to compare activation states of signaling cascades as well as expression levels of tumor and metastasis marker proteins. The results allowed us to cluster relapsed vs cured patients via differential activation states of several signal transduction pathways, such as MAPK and NFkB signaling. Based on this, we extracted a signature of eight proteins and protein modifications whose differential expression was sufficient to clearly distinguish relapsed from cured patients, and which therefore represent promising biomarker candidates.
As such, the results of this study demonstrate the power of multiplex protein profiling in general and of our novel DigiWest in particular for identification of prognostic markers for chemotherapy, through a direct and comprehensive analysis of protein expression and protein activation states.