143 MET, ECD shedding, and loss of E-cadherin scored all three markers. A third observer (S.M.W.) revised the scores. In case of disagreement, reevaluation was performed by M.J.D.H. and S.M.W. simultaneously until agreement was reached. Well-differentiated cancer cells that show no nuclei were omitted during scoring. Scoring of D1C2, A2H2-3 and E-cadherin immunoreactivity across WTSs Membranous immunoreactivities obtained using D1C2, A2H2-3 and NCH-38 differ markedly not only between, but also within slides. Staining intensities varied from 0 to 3 (17) and were either constant across cancer fields or varied between the center and periphery of cancer fields. To characterize and organize the observed interslide and intraslide variation, staining intensities were dichotomized and assessed for both the center and periphery of cancer fields. Cancer cells showing no (0) to weak (1) basal and/or lateral membranous immunoreactivity were assessed as negative, while cancer cells showing moderate (2) to strong (3) basal and/or lateral membranous immunoreactivity were assessed as positive. The periphery of cancer fields was defined as the outer 2-3 cell layers of a cancer field. The center of cancer fields was defined as other than the outer 2-3 cell layers of a cancer field. To consistently assess the possible combinations of central and peripheral scores, a two-dimensional scoring system was designed that describes four staining patterns: uniform negative, gradient toward the periphery, uniform positive and gradient toward the center (Figure 1, Supplementary figure 1). Using this system, the percentage(s) of the observed staining pattern(s) was scored per cancer. Scoring of MET protein status and ECD shedding across WTSs All cancers – scored for D1C2 and A2H2-3 – were assigned to one of three categories: MET negative, MET decoy receptor, or TM MET with or without the ECD. Analogous to previous work (17, 18), negative for MET was assigned when >90% of cancer cells showed absence of membranous immunoreactivity for C- and N-terminal MET. MET decoy receptor was assigned if the cancer cells showed more N-terminal than C-terminal membranous MET immunoreactivity in the form of the gradient toward the periphery and/or uniform positive staining pattern. The percentage of cancer cells showing the decoy receptor was calculated for each staining pattern by subtracting the percentage of C-terminal MET immunoreactivity from the percentage of N-terminal MET immunoreactivity. TM MET subjective or not subjective to shedding was assigned if the cancer cells showed more amounts of C-terminal or equal amounts of C-terminal than N-terminal membranous MET immunoreactivity in the form of the gradient toward the periphery and/or uniform positive staining pattern. 5
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