M7259

Quantification of immune cells (T cells, cytotoxic T cells, macrophages, plasma cells, and neutrophils) was assessed by IHC staining on tissue microarrays and automatic quantification by QuPath^{63 (link)}. CD45Ro (Ventana; 790-2930; Mouse monoclonal (UCHL-1)) for total immune cells, CD3 (Ventana; 790-4341; Rabbit monoclonal (2GV6)) for total T cells, CD8 (NOVO; PA0183; Mouse monoclonal (4B11)) for cytotoxic T cells, Foxp3 (Abcam; ab20034; Mouse monoclonal (236A/E7)) for Tregs, CD68 (DAKO; M0814; Mouse monoclonal (KP1)) for macrophages, CD163 (NOVO; NCL-CD163; Mouse monoclonal (10D6)) for M2 macrophages, MUM1 (DAKO; M7259; Mouse monoclonal (MUM1P)) for plasma cells, and MPO (DAKO; A0398; Rabbit polyclonal) for neutrophils were used as cell type markers. Four-μm-thick glass slides were stained using Ventana BenchMark XT and OptiView universal DAB staining kit (Ventana #760-700). Staining procedure of automatic stainer is antigen retrieval (100 °C for 24 min in citrate buffer), peroxidase inhibition (37 °C for 4 min in 3% H₂O₂), primary antibody (37 °C for 16 min), Linker (HQ linker; 37 °C for 8 min), polymer amplification (HRP multimer; 37 °C for 8 min), chromogen by DAB (37 °C for 8 min), counterstaining by hematoxylin (37 °C for 8 min), and post counterstain (37 °C for 4 min).

All cases were histologically confirmed to be diffuse large B-cell lymphomas according to the WHO Classification (2021). Histological sections were reviewed, and all cases were immunohistochemically stained for CD20 (M0755; Dako, Glostrup Kommune, Denmark), CD10 (NCL-L-CD10-270, Novocastra, Newcastle upon Tyne, UK), BCL6 (NCL-L-Bcl-6-564, Novocastra), and MUM1/IRF4 (M7259; Dako). Immunohistochemical staining was performed on a Ventana BenchMark ULTRA Immunostainer. They were categorized according to Hans classification into germinal center B-like (GCB) or non-germinal center B-like (non-GCB) immunophenotypes [33 (link)]. EBER in situ hybridization (EBER-ISH) was carried out in all cases using commercial reagents and the Inform EBER probe (800–2842, Ventana Medical Systems, Oro Valley, AZ, USA).