108 Chapter 4 Table 4: Multivariable analysis of disease-free survival and overall survival for patients having cancers showing transmembranous C-terminal MET with or without the ectodomain. Variable Disease-free survival Overall survival HR 95% CI P value HR 95% CI P value Age at diagnosisa 1.42 1.11–1.81 0.005 1.28 0.99–1.66 0.058 pT ≥ 1 1.25 0.52–3.03 0.616 2.66 0.81–8.77 0.109 pN ≥ 2 1.09 0.54–2.21 0.815 1.55 0.76–3.14 0.226 Extranodal extension present 3.80 1.79–8.04 0.000 2.50 1.25–5.00 0.010 Poor-undifferentiated opposed to well-moderate 1.03 0.54–1.93 0.937 1.64 0.87–3.08 0.126 Shedding in ≥35% of cancer cells 2.41 1.35–4.30 0.003 1.64 0.92–2.94 0.095 aThe HR was based on 10 year intervals Discussion The results presented here illustrate that A2H2-3 is specific in the detection of N-terminal MET under nonreducing and formalin-fixed paraffin-embedded conditions and is particularly sensitive using immunohistochemistry. ELISA is the most sensitive method to detect ectodomain shedding in vitro and shows that all investigated oral squamous cell carcinoma cell lines shed their N-terminus. Staining of C- and N-terminal MET using formalin-fixed paraffin-embedded parallel whole tissue sections shows that MET ectodomain shedding occurs in oral squamous cell carcinoma, which is associated with poor survival. In spite of the finding that ≤2% of average N-terminal MET immunoreactivity is associated with poor disease-free survival, the prognostic relevance of this result was not further pursued because such a low threshold is impractical in a diagnostic setting. It was decided to use western blotting to demonstrate the occurrence of ectodomain shedding in fresh frozen tissues, because the results show which—potentially oncogenic (21, 27)—C-terminal transmembrane-associated fragments are present. Although immunoreactivity of b-actin was low in about a quarter of the samples (Fig. 4), they were not excluded because b-actin can vary depending on grade and composition of the tumor (30, 31). Since other traditional reference genes, e.g., GAPDH, used as normalizers for mRNA expression data have also been described to show variable gene expression levels across cancers, we decided not to switch to another internal positive control protein (32). Verifying the absence of D1C2 immunoreactivity using ELISA on harvested culture media of the examined oral squamous cell carcinoma cell lines, could have been an additional confirmation of the occurrence of ectodomain shedding. As it is strongly believed that the totality of the presented
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