Multiplex IHC
tumour, HT, necrosis
SECONDARY, TUMOUR, NECROSIS
Sequential chromogenic multiplex IHC for vimentin/pancytokeratin (panCK), as previously described, was performed on tumour sections from patients included in the NP137 clinical trial and that were collected at C1D1 and C3D1. Dewaxed 4-μm-thick, paraffin-embedded tissue sections were subjected to two successive steps of IHC on a Ventana discovery XT platform (Ventana, Roche Diagnostics) using the REDMap and DABMap detection systems according to the manufacturer’s recommendations. In a first step for vimentin expression, slides were incubated with mouse monoclonal anti-human vimentin for 1 h (mouse, clone V9, Leica, no. NCL-L-VIM-V9, dilution 1:100) and incubated with rabbit monoclonal anti-mouse secondary antibody for 20 min (clone M1gG51-4, abcam, no. 125913, dilution 1:750). The slides were then incubated with biotinylated anti-rabbit secondary antibody for 24 min (Vector Laboratories, dilution 1:200) followed by the addition of the streptavidin–alkaline phosphatase complex. Immunostaining was detected by incubation with naphthol and Fast red. Tissue sections were counterstained with Gill’s haematoxylin, dehydrated and mounted. Whole histological slides were digitized at ×20 magnification using a Hamamatsu 2.0 HT scanner. After removal of coverslips, slides were incubated in 100% ethanol until complete erasure of red colour. In a second step, to show panCK expression, the slides were incubated with mouse monoclonal anti-panCK antibody for 1 h (mouse, clone CKAE1AE3, Dako Belgium, no. M351529-2, dilution 1:150) and then with rabbit monoclonal anti-mouse secondary antibody for 20 min. The slides were then incubated with biotinylated anti-rabbit secondary antibody for 28 min (Vector Laboratories, dilution 1:200) followed by the addition of streptavidin–alkaline phosphatase. PanCK immunostaining was detected by incubation with naphthol and Fast red. The IHC slides were counterstained with Gill’s haematoxylin, dehydrated, mounted and again digitized. Image processing and analysis for Hynrid EMT score computation were performed using Visiomorph DP 2018.4 to determine vimentin and panCK co-expression in each tissue slide. Briefly, each pair of vimentin- and panCK-labelled virtual slides, which were acquired from the same tissue section, was subjected to image registration. Vimentin- and panCK-positive areas were automatically detected in the aligned virtual slides to evidence their co-expression in tumour cells. This co-expression was evaluated on whole slides at ×10 magnification to take into account potential imperfections. Manual corrections were carried out to exclude irrelevant sample parts, such as necrosis. Cell nuclei negative for both markers were also excluded, to focus only on cytoplasmic areas where colocalization could occur.