42 Chapter 3 Deregulated HGF/SF-MET signaling has been implicated in many human solid cancers (19), which has led to the development of biologicals that target MET (19). Today’s challenge lies in the reliable stratification of patients eligible for treatment with MET inhibitors (19). Although MET is abundantly expressed and acts as an orchestrator of invasive growth in HNSCC (20), its role as a prognostic factor remains unclear (21-30). This might be due to the use of a variety of antibodies resulting in varying staining patterns and scoring systems. Moreover, several antibodies showed significant lot-to-lot variability in terms of specificity, sensitivity and staining patterns (31, 32). Using the Rimm Lab Algorithm for antibody validation (33) as guidance, this study investigates the specificity and sensitivity – for single lots – of five commercial antibodies directed against the C-terminus of MET (i.e., D1C2, CVD13, SP44, C-12 and C-28) under reducing, native and formalin-fixed paraffin-embedded (FFPE) conditions using a panel of well characterized cell lines – of which one was silenced for MET using a siRNA. Next, the antibodies that behaved reliably across all examined conditions (i.e., D1C2 and CVD13) were used to explore MET immunoreactivity across whole tissue sections of a selection of oral SCC. Finally, using the antibody that is most sensitive in the detection of membranous MET (i.e., D1C2), it was examined whether MET immunoreactivity is associated with the survival of 179 patients diagnosed with oral and oropharyngeal SCC of whom long-term clinico-pathological follow-up was available. Materials and methods Antibodies Western blot analysis, immunocyto- and immunohistochemistry were performed using five commercial antibodies directed against the C-terminus of MET, specifically D1C2 (Cell Signaling Technology®; Leiden, The Netherlands), CVD13 (Life Technologies™/Invitrogen™; Bleiswijk, The Netherlands), SP44 (Spring™ Bioscience; Pleasanton, CA 94566, USA), C-12 and C-28 (Santa Cruz Biotechnology, Inc.; Heidelberg, Germany). Additional property information on the antibodies is indicated in Table 1.
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