61 MET immunoreactivity and poor prognosis Under native conditions, MET immunoreactivity – obtained with D1C2 and CVD13 – was observed in the nucleus, in the cytoplasm and at the membrane. This finding is not unexpected since MET and/or MET C-terminal fragments have been observed at each of these cellular locations (47-50). In contrast, MET immunoreactivity was only observed in the cytoplasm and at the membrane of FFPE cells indicating that the nuclear epitope cannot be detected by these two antibodies under FFPE conditions. When comparing the membranous and cytoplasmic immunoreactivities obtained under FFPE conditions, the observed intensities indicate that CVD13 was most sensitive in the detection of cytoplasmic MET and D1C2 was most sensitive in the detection of membranous MET in FFPE cancer cells and tissues. Because antibodies as well as a selection of tyrosine kinase inhibitors can target the receptor when it is located at the membrane (19), membranous MET immunoreactivity was further investigated in a series of oral and oropharyngeal SCC using D1C2. It was observed that membranous MET immunoreactivity is either constant across the cancer (uniform negative or positive staining) or differs between the tumor center and periphery (variable staining). Before discussing the results obtained with survival analysis, we would like to stress that we realize that using boundaries for survival analysis based on biology is not indisputable. Therefore, efforts are needed – in the future – to validate the relation between MET staining patterns and survival. Hopefully, such efforts will result in a standardized scoring system for MET immunoreactivity that is applicable in a routine diagnostic setting. Univariable survival analysis revealed that patients with cancers showing the variable staining pattern perform significantly better than patients showing either uniform staining patterns (negative or positive) in terms of 5-year OS and DFS. The significant association between uniform positive MET staining and poor survival is not unexpected, because MET is a known orchestrator of invasive growth (18, 20, 51, 52). Although counterintuitive, the significant association between uniform negative MET staining and poor survival is not illogical as MET is expressed on the surface of epithelial cells under physiological conditions (18), which is corroborated by our results showing strong MET immunoreactivity with salivary glands ducts. Therefore, it is hypothesized that a tight balance exists between the amount of MET protein and tissue homeostasis. The observation that both low and high expression levels of ERBB2 – also a RTK involved in many epithelial cancers (53) – are associated with 3
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