IHC staining was performed to evaluate the expression level of MYC, BCL2 or BCL6 proteins in our DLBCL samples, using 3 µm formalin-fixed paraffin-embedded (FFPE) tissue sections which were subjected to 3 min. Ethylenediaminetetraacetic acid (EDTA) buffer-based heat-induced antigen retrieval. The samples were probed with monoclonal primary antibodies against CD10 (clone 56C6; Cat# MA5-14050, 1:5, ThermoFisher Scientific, Carlsbad, CA, USA.), IRF4/MUM1 (clone EP190; Cat# BSB-6958, 1:80, BioSB, Santa Barbara, CA, USA), BCL2 (RTU clone SP66; Cat# 790-4604, Roche Tissue Diagnostics, Oro Valley, AZ, USA), MYC (clone EP121; Cat# BSB-6581, 1:30, BioSB), BCL6 (RTU clone GI191E/A8; Cat# 760-4241, Roche Tissue Diagnostics), and Ki67 (clone SP6; Cat# MA5-14520, 1:200, ThermoFisher Scientific). All staining was performed using the Ventana benchmark ULTRA IHC staining module (Ventana, Tucson, AZ, USA). As previously described [19 (link)], for COO determination based on Hans’ algorithm, positivity cut-off for CD10, IRF4/MUM1, and BCL6 expression was ≥30% stained cells, while the expression positivity cut-off for BCL2 or MYC was ≥50% or ≥40% of stained cells, respectively.
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