Cd30 ber h2

FFPE sections were subjected to immunoperoxidase analysis with monoclonal antibodies as follows: CD2, CD4, CD5, and CD56 (Novocastra Laboratories, Newcastle, UK); CD3, CD8, UCHL‐1/CD45RO, L26/CD20, Ber‐H2/CD30, and ALK1 (Dako, Santa Clara, CA); βF1 (T‐cell receptor [TCR] β chain; T Cell Science, Cambridge, MA); TCR 1153 (TCR‐γ; clone γ 3.20) and TCRδ constant region (clone 5A6.E9; Thermo Fisher Scientific),23 TIA‐1 (Coulter Immunology, Hialeah, FL), granzyme B (Monosan, Uden, the Netherlands), PD‐L1 (clone SP142; Spring Bioscience, Pleasanton, CA), and ALK 5A4.28 The reactions were considered positive with a cut‐off of 30% (Figure 1D‐F). Tumor cell and microenviroment PD‐L1 expression was considered positive when ≥10% of the tumor cells and nonmalignant stromal cells showed membranous and/or cytoplasmic PD‐L1 staining, resepectively.29, 30To evaluate the presence of EBV small ribonucleic acids, we subjected formalin‐fixed, paraffin‐embedded sections to in situ hybridization using EBV‐encoded small nuclear early region (EBER) oligonucleotides, as previously reported.8

FFPE sections were subjected to immunoperoxidase analysis with the following monoclonal antibodies: CD4, CD5, and CD56 (Novocastra Laboratories, Newcastle, United Kingdom); CD3, CD8, CD10, L26/CD20, Ber-H2/CD30, B-cell lymphoma 6 (BCL6), and 22C3/PD-L1 (Dako, Santa Clara, CA); CXCL13 (R&D Systems, Minneapolis, MN); granzyme B (Monosan, Uden, The Netherlands); SP98/inducible T-cell costimulator (ICOS) and γ 3.20/ T-cell receptor γ (TCR-γ; Thermo Fisher Scientific); programmed cell death protein 1 (PD-1; Abcam, Cambridge, United Kingdom); βF1 (TCR β chain; T Cell Science, Cambridge, MA); H-41/TCRδ (Santa Cruz Biotechnology, Dallas, TX); and T-cell intracellular antigen 1 (TIA-; Coulter Immunology, Hialeah, FL). The reactions were considered positive with a cutoff of 30%. Neoplastic PD-L1 expression (nPD-L1) was considered positive if ≥10% of the tumor cells had membranous and/or cytoplasmic PD-L1 staining. A case was considered positive for PD-L1 in the microenvironment when ≥20% comprised nonmalignant cells with moderate or strong membrane or cytoplasmic PD-L1–specific staining, among the total tissue cellularity.¹³ (link)
We subjected FFPE sections to ISH using EBER oligonucleotides as previously reported to evaluate the presence of EBV small ribonucleic acids.³ (link)

mIHC was performed using an Opal automation Multiplex IHC kit (PerkinElmer NEL801001KT or NEL821001KT, or equivalent) on Leica BOND Rx platform followed by IF 6-colorWJJ-CD30 protocol in CAP-controlled area within the Oncology and Immunology Unit of WuXi AppTec. Human FFPE specimens were labeled with different primary antibodies (CD30 Ber-H2, Dako M0751; FcγRΙ⁺ OTI3D3, Abcam ab140779; CD68 KP-1, Ventana 790–2931; PD-L1 SP263, Ventana 790–4905; CD8 SP57, Ventana 790–4460), followed by appropriate secondary antibodies (Polymer HRP from Opal automation Multiplex IHC kit) and different Opal dyes, and finally counterstained with DAPI (spectral 4′,6-diamidino-2-phenylindole), rabbit immunoglobulin G (IgG; Abcam ab172730, EPR25A), and mouse IgG1 (Abcam ab18443, kappa monoclonal MOPC-21) were used as isotype control. Whole slide images were acquired for each case using a Leica Aperio VERSA 8 automated microscope. Image analysis was performed using the HALO software package (Indica Labs), and segmentation and mark-up of individual cells were performed, reviewed, and scored blinded by two pathologists using the IndicaLabs-HighPlex FL module.