Pannoramic 1000 digital slide scanner

The prostate specimen was fixated in formalin for at least 24 h. After, fixation specimens were sectioned from apex to base in 4-mm slices using a TruSlice specimen cut-up system (Cellpath Ltd, Newtown, UK). The prostate slices were fitted in cassettes, embedded in paraffin, and cut into whole-mount pathology slides (4 μm thick).
Whole-mount pathology slides were scanned on high resolution (40× enlargement, 20× objective, 2.1 camera lens) using a Pannoramic 1000 Digital Slide Scanner (3DHISTECH, H-1141 Budapest, Öv u. 3., Hungary) and uploaded to a web-based pathology annotation tool (Slidescore, Amsterdam, the Netherlands). The parasagitally cut apical and basal pathology slides were not included for annotation in the current study.

Based on the results of the NanoString assay, we performed immunohistochemistry (IHC) for LAG‐3 (clone EPR4392; Abcam) and PD‐L1 (clone SP142; Ventana). All slides were scanned using a Pannoramic 1000 digital slide scanner (3DHistech). An INFINITT DPS (INFINITT Healthcare) was used as the image viewing system. Images with an area of 0.37 mm² were captured for two representative areas to measure the degree of infiltration of LAG‐3‐positive cells in tumors. The number of positive cells and the ratio (%) of positive cells to total cells in the captured images were investigated using QuPath software.²³ A combined positive score (CPS) (the number of positive tumor cells, lymphocytes and macrophages divided by the total number of viable tumor cells, multiplied by 100) system was used to evaluate PD‐L1 expression. One pathology resident (HL) and one hematopathologist (JC) independently measured the CPS of all PD‐L1 stains, and the scores of cases with discrepancies were established by consensus.

All 1472 HE slides were digitized at the Radboud University Medical Center (RUMC) using a Pannoramic 1000 digital slide scanner (3DHistech ltd., Budapest, Hungary) at a resolution of 0.24 μm/pixel. Scans were pseudonimized by the RUMC and researchers remained blinded throughout the annotation- and validation process.