Mum1 clone ma695

Immunohistochemistry (IHC) was performed using antibodies against MYC (clone EP121, Cell Marque, Rocklin, CA, USA), BCL2 (clone 124, DAKO, Carpinteria, CA, USA), BCL6 (clone LN22, Novocastra, Newcastle, United Kingdom), CD10 (clone 56C6, Novocastra), and MUM1 (clone Ma695, Novocastra). IHC staining was performed using the Ventana Benchmark XT system (Ventana Medical Systems, Tucson, AZ, USA) or a Bond-Max automated immunostainer (Leica Microsystems, Melbourne, Australia). Cell of origin was assessed according to the Hans criteria [11 (link)]. IHC score was determined to be the percentage of tumor cells with robust immunostaining evaluated by 10 % increments. Cutoff values (i.e. IHC scores) of ≥40 % for MYC, ≥30 and ≥60 % for BCL2 and ≥50 % for BCL6 were determined to have discriminant prognostic power based on the receiver operator characteristic (ROC) curves and were thus used for classifying cases into MYC-, BCL2- or BCL6-negative and-positive groups.

IHC was prospectively performed on the routine diagnostic basis using representative whole formalin-fixed paraffin-embedded (FFPE) tissue sections and antibodies against CD3 (clone 2GV6; Ventana Medical Systems, Tucson, AZ, USA), CD20 (clone L26; DAKO, Carpinteria, CA, USA), BCL2 (clone 124; DAKO, Carpinteria, CA, USA), BCL6 (clone LN22; Novocastra, Newcastle, UK), CD10 (clone 56C6; Novocastra), MUM1 (clone Ma695; Novocastra), MYC (clone EP121; Cell Marque, Rocklin, CA, USA), and Ki-67 (clone MIB-1; DAKO). Staining was performed using a Ventana Benchmark XT (Ventana Medical Systems) or a Bond-Max autostainer (Leica Microsystems, Melbourne, Australia). COO was determined using the IHC-based Hans algorithm as previously described [29 (link)]. DE status was defined as the co-expression of MYC (in ≥40% of tumor cells) and BCL2 (in ≥70% of tumor cells) as previously described [12 (link)]. TMA was constructed using the FFPE tissue blocks from 50 selected cases with DLBCL, and we performed MYC, BCL2, BCL6 FISH on the TMA. FISH was performed using Vysis LSI BCL2 Dual Color Break Apart Rearrangement Probe (Vysis, Downers Grove, IL, USA), Vysis LSI BCL6 Dual Color Break Apart Rearrangement Probe (Vysis), and Vysis LSI MYC Dual Color Break Apart Rearrangement Probe (Vysis)).