Immunohistochemical Profiling of Tumor Markers

Immunohistochemical staining was performed using the streptavidin–biotin method^{11 (link)}, with antibodies to the following: pan-TRK (clone: EPR17341; Abcam, Cambridge, MA, USA) as previously described^{3 (link)}. IHC for p53 (Clone: DO-7; DAKO, Glostrup, Denmark), Ki-67 (Clone: MIB-1; DAKO, Glostrup, Denmark), and β-catenin (Clone: Rabbit Poly; DAKO, Glostrup, Denmark) were also performed. The diffuse staining pattern was judged as positive for pan-TRK, regardless of the staining intensity on TMA-based IHC. Three positive cases for pan-TRK IHC on TMA sections were also stained on the whole FFPE sections. IHC for p53 was performed on TMA sections, while IHC for β-catenin and Ki-67 was performed and evaluated using whole sections for only 3 positive cases. The p53 IHC findings were classified into overexpression, wild-type, and complete absence^{15 (link),16 (link)}. The Ki-67 labeling index was evaluated in representative areas that showed the highest immunoreactivity by counting the number of positive cells among 1000 tumor cells.