109 MET ECD shedding and poor DFS data are supportive for the occurrence of ectodomain shedding in vitro, such an experiment was not performed. When comparing C-terminal MET immunoreactivities obtained using western blotting and immunohistochemistry, discrepancies are observed (positive versus negative, Supplementary Fig. 4). This probably has to do with only scoring membranous immunoreactivity under formalin-fixed paraffin-embedded conditions. Absence of membranous immunoreactivity does not imply absence of cytoplasmic immunoreactivity (Fig. 8) representing either the precursor, the mature receptor and/ or C-terminal fragments (17). Discrepancies between reducing and formalin-fixed paraffin-embedded conditions can also be explained by tumor heterogeneity. The cells sampled in the fresh frozen tissues may represent a different clone of cancer cells than the cells sampled in the formalin-fixed paraffin-embedded tissues of corresponding cancers. Tumor heterogeneity can also explain the observation that two cancers were scored negative under reducing conditions and positive using immunohistochemistry (Fig. 8). Another possible explanation might be differences in histopathology of the examined oral squamous cell carcinoma. However, we find the number of included samples too low to perform such analyses. Fig. 8: 4 × 4 matrix illustrating agreement and discrepancies for D1C2 −/+ immunoreactivity under reducing and formalin-fixed paraffin-embedded conditions (×5 objective). a Combinations of D1C2−/+immunoreactivity observed under reducing versus formalin-fixed paraffin-embedded conditions. Illustrated with photographs of observed D1C2 immunoreactivities in four examined formalin-fixed paraffin-embedded cancers. b Photographs of parallel sections stained with A2H2-3 of the same cancer regions depicted under a showing that both stainings can behave similarly. 4
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