Pannoramic 1000 scanner

Formalin fixed paraffin-embedded (FFPE) tumour samples were sectioned and stained with haematoxylin and eosin (H&E) before pathological review and determination of tumour purity. Sections were also stained with anti-smooth muscle actin (Clone E184, Abcam), anti-desmin (Polyclonal, Abcam), anti-Ki67 (MIB-1, Dako), and anti-PAX8 (polyclonal, Proteintech) using the Ventana BenchMark Ultra fully automated staining instrument (Roche Diagnostics, USA). H&E and IHC slides were digitally scanned (20 × magnification) using the Pannoramic 1000 scanner (3DHISTECH Ltd.). High-definition images were uploaded into CaseCenter (3DHISTECH Ltd.), and images were processed using Adobe Illustrator.

All stained sections were automatically scanned using a Pannoramic1000 scanner (3DHISTECH, Budapest, Hungary) with an AdimecQ camera (Adimec, Woburn, MA, USA) at standard settings. Digital images were visualized using CaseViewer-2.3 (3DHISTECH, Budapest, Hungary) and exported as TIFF files. For each image, the regions of interest (ROI) were determined as described [28 (link)]. In short, one central wound region of 700 µm was demarcated and the muscle layer was digitally isolated from the image. In the α-SMA staining, the salivary glands and the large blood vessels were excluded from the analysis. An Image J (Fiji, NIH-LOCI, Milwaukee, WI, USA) macro for each staining was applied as described [28 (link),32 (link),33 (link)]. The area measurements are expressed as the mean percentage ± SD of the ROI in mm².

FGFR2 protein level was examined using immunohistochemistry and quantified in digitalized slides as described previously [20 (link)]. Immunohistochemical staining (IHC) was conducted on 5‐μm paraffin tumour sections using a mouse monoclonal anti‐FGFR2 antibody (H00002263‐M01; clone 1G3, Abnova, Taipei City, Taiwan). All slides were digitalized (Pannoramic 1000 Scanner, 3DHistech, Sysmex, Kobe, Japan) for quantification in 0–300 H‐score scale. Cases from 1st tercile of H‐score were regarded as FGFR2low and cases from 2nd and 3rd terciles were classified as FGFR2high.

The stained TMA slides were scanned using the Pannoramic 1000 scanner (3DHistech, Budapest, Hungary) and visualized using the CaseViewer software (Version 2.4; 3DHistech, Budapest, Hungary). The evaluation was performed by a board-certified pathologist (R.E.) blinded to clinicopathological and outcome data visualizing H&E, CD3, and TIGIT scans of each TMA core next to each other. First, H&E slides, stained according to the standard in-house protocol, were reviewed regarding the morphology and growth pattern of OSCC. For each case, the semiquantitative percentage of TIGIT expression within CD3+ T cells was assessed (ranging from 0–100%). Moreover, TIGIT assessment was separately performed for the stromal and intraepithelial tumor areas, respectively.

Immunohistochemical staining (IHC) for FGFR2 in all tumors was conducted using a mouse monoclonal anti-FGFR2 antibody (H00002263-M01; clone 1G3, Abnova, Heidelberg, Germany) (Figure 2). To confirm specificity of the staining, additional IHC with a mouse anti-FGFR2 antibody (Sc-6930, Santa Cruz, CA, USA) was performed in randomly selected samples. Following manufacturer’s recommendations, tissue samples of gastric adenocarcinoma and lymph node were used as positive and negative controls for IHC, respectively. Immunohistochemical procedures were carried out on 5-µm paraffin sections, as reported previously [9 (link),15 (link)]. All slides were digitalized using Pannoramic 1000 Scanner (3DHistech, Sysmex, Kobe, Japan). FGFR2 levels were quantified according to the semiquantitative H-score approach by two independent pathologists (MB, HR). The data were presented in 0–300 scale resulting from multiplication of percentage of positive cells by intensity of staining: 0—no staining, 1–3—increased intensity of both cytoplasmic and membrane staining (subgroups by H-score: 0–75 for negative/weak; 76–150 for moderate; 151–225 for strong; 226–300 for very strong expression) (Figure 2). Cases from 1st tercile of H-score were regarded as FGFR2low and cases from 2nd and 3rd terciles were classified as FGFR2high.

The formalin-fixed, paraffin-embedded (FFPE) samples were stained for both PD-L1 and CD8. Immunohistochemistry of the FFPE tumor samples was performed on a BenchMark Ultra autostainer (Ventana Medical Systems). Briefly, paraffin sections were cut at 3 µm, heated at 75 °C for 28 min and deparaffinized in the instrument with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 32 min at 95 °C (CD8) or 48 min at 95 °C (PD-L1). CD8 was detected using clone C8/144B (1/100 dilution, 32 min at 37 °C, Agilent/DAKO) and PD-L1 was detected using clone 22C3 (1/40 dilution, 1 h at RT, Agilent/DAKO). Bound antibody was detected using the OptiView DAB Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems). A Pannoramic® 1000 scanner from 3DHISTECH was used to scan the slides at a 40 × magnification. The stained FFPE slides were scored by a blinded pathologist using Slidescore (www.slidescore.com). Of each biopsy, five representative areas of 0.2mm² were selected to assess the number of CD8⁺ cells/mm².