Epr17341

IHC was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections with a thickness of 4 µm using an automated staining instrument (BENCHMARKXT, Roche, Tucson, AZ, USA). Primary antibodies against CD117 (YR145, maxim-BIO, Fuzhou, China), DOG-1 (OTI1C6, ZSGB-BIO, Beijing, China), CD34 (EP88, ZSGB-BIO, Beijing, China), SDHB (MAB-0736, maxim-BIO, Fuzhou China), BRAF V600E (H03529, Roche Diagnostics, Tucson, AZ, USA), and Pan-TRK (EPR17341, Roche Diagnostics, Monza, Italy) were used. The testing and assessment were performed according to the manufacturer’s instructions for every biomarker. For Pan-TRK, nuclear, cytoplasmic, or membranous staining in more than 5% of tumor cells was considered positive [36 (link)]. All IHC-stained sections were interpreted by two experienced pathologists.

FFPE TMA blocks were cut in 4 µm-thick sections and placed on slides. IHC staining for pan-TRK expression was performed on the Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ) with iVIEW DAB Detection Kit (Ventana Medical Systems, Tucson, AZ), using a commercially available pan-TRK assay (rabbit monoclonal antibody, clone EPR17341, Assay, RTU, Roche, Ventana). Normal appendix and brain tissues were used as positive controls. The immunohistochemical evaluation included (i) location (membranous, cytoplasmic, nuclear, mixed), (ii) intensity (strong, moderate, weak, and negative) and (iii) the extent (percentage of positive tumor cells) of the IHC staining. Membranous, cytoplasmic, and nuclear staining patterns were considered positive if ≥1% of tumor cells exhibited positivity at any intensity above background [29 (link)]. Diffuse staining was defined as moderate to strong expression in ≥50% of tumor cells [39 (link)].

NTRK immunohistochemical staining was performed using the antibody clone EPR17341 (Roche, Tusan, United States) to assess NTRK1, NTRK2, and NTRK3 protein expression in the FFPE samples. Positive results were defined as staining above background in at least 1% of tumor cells in any pattern, including membranous, cytoplasmic, perinuclear, or nuclear.