Pan trk

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.

Tumor and liver tissues were fixed in 4% phosphate-buffered formalin and embedded in paraffin using standard procedures. Tumor organoids were released from BME2 by incubation in Dispase II (Sigma-Aldrich, Cat. No. D4693). Organoids were fixed in 4% phosphate-buffered formalin in PBS for 30 min at room temperature following encapsulation in HistoGel (Thermo Fisher Scientific, Cat. No. HG-4000-012) and subsequent dehydration and paraffin embedding.
Histopathological evaluation was assessed by three board-certified pathologists (MSM, JV and LMT). Tumors were classified based on architecture and cytological features, and graded according to the Edmondson grading system^{10 (link),11} . The following primary antibodies were used for automated diagnostic immunohistochemical staining on a Benchmark XT device (Ventana Medical Systems) at the Institute of Pathology of the University of Basel: AFP (Ventana, Ref-Nr. 760-2603), ARG1 (Ventana, Ref-Nr. 760-4801), CD10 (Ventana, Ref-Nr. 790-4506), CD56 (Ventana, Ref-Nr. 790-4465), CHGA (Ventana, Ref-Nr. 760-2519), GPC3 (Ventana, Ref-Nr. 790-4564), HLA-ABC (Abcam, Cat. No. ab70328), Hep Par-1 (Ventana, Ref-Nr. 760-4350), KRT19 (Ventana, Ref-Nr. 760-4281), Ki-67 (Dako, Cat. No. IR626), Pan-TRK (Abcam, Cat. No. ab181560), PD-L1 (Ventana, Ref-Nr. 740-4907), SYP (Ventana, Ref-Nr. 790-4407), and SSTR2 (Abcam, Cat. No. ab134152).