234 Chapter 8 it was decided not to examine overall average MET immunoreactvity per tumor. Instead, average MET immunoreactivity in the periphery versus average MET immunoreactivity in the center of the tumor was investigated because, in the majority of cancers, wildtype MET is transcriptionally activated by stimuli originating from the reactive stroma such as hypoxia, inflammatory cytokines, stromal HGF, and pro-angiogenic factors (3, 27-29). To accomplish this peripheral versus center examination, MET immunoreactivity was examined by means of a scatter plot based analysis, in which each data point represents the pattern of MET immunoreactivity across a cancer section (chapters 3 and 4). For D1C2 immunoreactivity it was observed that data points are scattered across the entire chart area, indicating that MET staining is either uniform (negative or positive), or variable across OSCC (chapter 3). Plotting survival status over the data points, revealed a higher concentration of events for OSCC with the uniform compared to the variable staining pattern. Visual examination of this difference in concentration of events, ultimately lead to the thresholds used for survival analyses, which revealed that both uniform staining patterns (negative and positive) are associated with poor prognosis. In contrast to D1C2 immunoreactivity, A2H2-3 immunoreactivity is generally low across cancers and shows no relationship with survival status (chapter 4). Alignment of average A2H2-3 versus average D1C2 immunoreactivity, again using the scatter plot based analysis, led to the stratification of OSCC into three categories based on MET protein status: MET negative (no MET immunoreactivity), decoy MET (more positivity for the N-terminal moAb), and transmembranous C-terminal MET (equal positivity for both moAbs (complete MET), or more positivity for the C-terminal moAb (MET-EC-) (chapter 4). Survival analyses showed that MET negative cancers are associated with poor prognosis for the entire sample population and that D1C2 uniform positivity, and ECD shedding are associated with poor prognosis for patients positive for transmembranous C-terminal MET immunoreactivity (chapters 3 and 4). Extrapolation of the MET staining patterns, defined using the TMA, to WTSs using a newly developed scoring system (chapter 5), revealed not only a substantially lower portion of MET negative cancers, but also a lack of association between MET negativity and poor survival. This observation was explained by tumor heterogeneity. Where WTSs show combinations of uniform (negative or positive) and variable (gradient towards the periphery or center) staining patterns, TMAs were
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