110 Chapter 4 Fig. 9: 4 × 4 matrix illustrating agreement and discrepancies for the presence of MET ectodomain shedding under reducing and formalin-fixed paraffin-embedded conditions (×5 objective). a Combinations of MET ectodomain shedding −/+ 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 illustrating that D1C2 and A2H2-3 immunoreactivity can be identical—first row—or that there can be MET ectodomain shedding—second row. Tumor heterogeneity is also a possible explanation for the inconsistencies observed between reducing and formalin-fixed paraffin-embedded conditions with respect to ectodomain shedding (Fig. 9). Therefore, we assume that both methods—[1] western blotting using D1C2 and fresh frozen tissues and [2] immunohistochemistry using D1C2 and A2H2-3 on parallel formalin-fixed paraffin-embedded sections—reliably depict the occurrence of MET ectodomain shedding. Since immunohistochemistry is widely used in routine diagnostics, we opted to use this technique to investigate whether there is an association between MET ectodomain shedding and survival. We have established previously—using the same tissue microarray—that membranous D1C2 immunoreactivity is either constant (uniform negative or positive staining) across or differs between the center and periphery (variable staining) of oral squamous cell carcinoma (n = 157) and HPV negative oropharyngeal squamous cell carcinoma (n = 22) (18). Univariable survival analyses revealed that both uniform negative (n = 41) and positive (n = 16) staining patterns perform significantly worse than the variable staining pattern (n = 122) in terms of disease-free survival (HR = 2.00; 95% CI, 1.29–3.08 and P = 0.002 for uniform negativity and HR = 1.92; 95% CI, 1.01–3.65 and P = 0.048 for uniform positivity) and overall survival (HR = 2.17; 95% CI,

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