Benchmark ultra instrument

Immunohistochemical (IHC) staining for BRAF^V600E and Ki67 were performed in 34 and 32 radiogenic, and in 38 and 36 sporadic MPTCs, respectively, for which the additional tissue sections were available.
IHC staining for BRAF^V600E was performed as described before (34 (link), 35 (link)). In brief, a mouse monoclonal anti-BRAF (mutated V600E) antibody (VE1) ab228461(Abcam) at a 1:100 dilution and the Novolink Polymer Detection System (250T) (Leica RE7140-K) were used to detect IHC reaction product. In our hands, the BRAF^V600E positivity on IHC was concordant with the presence of the BRAF^V600E mutation (36 (link)).
The proliferative activity of tumors was evaluated by IHC using a Ki67 antibody (clone MIB-1; DAKO, Glostrup, Denmark, 1:100 dilution) in a Ventana BenchMark ULTRA instrument. The Ki67 labeling index (Ki67 LI) was determined with the image-analyzing software (CountσCell, Ki67 antigen Semi-Auto Counter, Seiko Tec LTD, Fukuoka, Japan) in a total of approximately 1,000 PTC cells per case (LZ). Image analysis was performed in a blind for the BRAF^V600E status manner.

Immunohistochemical (IHC) staining for BRAF^V600E (LZ, TIR) was performed as described before (33 (link)). In brief, we used a mouse monoclonal anti-BRAF (mutated V600E) antibody (VE1) ab228461(Abcam) at a 1:100 dilution and the Novolink Polymer Detection System (250T) (Leica RE7140-K) to detect IHC reaction product. IHC staining was evaluated by three qualified observers (L.Z., T.B., T.I.R.), and full agreement was achieved; there were no specimens interpreted by any observer as potentially false-negative or false-positive. A close correlation between the results of the VE1-based IHC for BRAF^V600E and molecular methods of the detection of the BRAF^V600E mutation at the DNA level has been reported in a meta-analysis (35 (link)) and confirmed in our previous study using formalin-fixed paraffin-embedded material (36 (link)). Therefore, we assumed the BRAF^V600E positivity on IHC was indicative of the BRAF^V600E mutation.
The proliferative activity of tumors was evaluated by IHC using Ki67 antibody (clone MIB-1; DAKO, Glostrup, Denmark, 1:100 dilution) in a Ventana BenchMark ULTRA instrument. The Ki67 labeling index (Ki67 LI) was determined with the image-analyzing software (CountσCell, Ki67 antigen Semi-Auto Counter, Seiko Tec LTD, Fukuoka, Japan) in a total of ~1,000 PTC cells (LZ). Image analysis was performed in a blind for the BRAF^V600E status manner.

This study was approved by the Institutional Review Board of the Ajou University School of Medicine (AJIRB-BMR-KSP-20-396). Informed consent was waived due to the retrospective study design. We retrospectively selected 57 patients with advanced NSCLC who received PD-1 inhibitors from 2016 to 2021. The patients’ clinicopathological characteristics are summarized in Supplementary Table S1. All patients underwent lung biopsy or surgical resection. Inoperable cases were treated with PD-1 inhibitors, and surgical cases were treated with PD-1 inhibitors for subsequent recurrence or metastasis. Among the 57 patients, 31 had LUAD and 26 had LUSC. Patients treated with PD-1 inhibitors were classified as responders (complete response or partial response) or non-responders (stable disease or disease progression) [11 (link)]. The PD-L1 immunohistochemistry (clone: SP263, rabbit monoclonal, Roche, Basel, Switzerland) was performed using the OptiView DAB Immunohistochemical Detection Kit on a Ventana BenchMark ULTRA instrument.

Immunohistochemistry was performed on formalin-fixed paraffin embedded 4 µm-thick tissues on a BenchMark Ultra instrument (Ventana). The primary antibody and dilution used in this study are as follows: rabbit monoclonal anti-ALK (clone D5F3; Cell Signaling Technology, Danvers, MA) applied at 1:50 and detected with Ultraview Universal DAB detection kit (Ventana). IHC pictures were taken using a Leitz DMRB microscope (Leica) and a DS-Ri1 camera (Nikon).

Consecutive 4-μm sections from formalin-fixed paraffin-embedded (FFPE) tumor tissues were prepared for immunohistochemistry (IHC) analysis. The Ventana FAP (SP325) Robust Prototype Assay (RPA) by Ventana Medical Systems Inc. (Tucson, AZ, USA) was used for the staining process. This assay, which uses a rabbit monoclonal antibody, clone SP325, obtained from Spring Biosciences (Pleasanton, CA, USA), was designed to detect Fibroblast Activated Protein (FAP) in FFPE samples. The staining was carried out using the OptiView DAB IHC Detection^TM kit on a VENTANA BenchMark ULTRA instrument. To evaluate the reactivity of the secondary antibody and detection chemistry, an immunoglobulin-matched rabbit monoclonal antibody (VMSI, Catalog No. 790-4795) was employed as a negative control. The staining intensity was scored manually on a semi-quantitative scale ranging from 0 (negative) to 3 (or “3+”) by a certified anatomic pathologist. The percentage of cells stained positively, covering both normal stroma and neoplastic cells, was recorded for each intensity level. An H-score from 0-300 was calculated by combining the stromal and tumor cell staining (FAP-intensity score). The stroma-tumor H-score incorporated components from both normal stromal and tumor staining intensities along with the percentage of positively stained cells.