Ish iview kit

In situ hybridization (ISH) was performed on the same FFPE blocks, which were used for immunohistochemistry. The EBER-1 dinitrophenyl (DNP) probe was used to detect the expression of EBER-1 system (ISH iView kit; Ventana Medical Systems Inc., Tucson, AZ, Cat# 760-097). EBER-ISH was performed using an automated Ventana BenchMark XT system (Ventana Inc.) in accordance with the manufacturer's protocol. Briefly, FFPE sections were treated with EZ Prep buffer (Ventana Inc.) to remove paraffin, rehydrated, and then digested with ISH protease 1 (Ventana Inc., Cat# 780-4147). EBER 1 DNP probe was then administered and allowed to hybridize, followed by stringency washes as per the manufacturer's instructions using SSC buffer (Ventana Inc., Cat # 950-110). Slides were counterstained with Red Counterstain II (Ventana Inc., Cat# 780-2218). Serial sections of all samples were also stained with oligo-T probes to ascertain RNA preservation in each sample. EBV-infected tissue from a tonsil classified as having infectious mononucleosis was used as a positive control for EBER-1. Tonsil tissue obtained from a healthy individual was used as a negative control.

The IHC staining markers included CD20, CD79a, PAX5, CD3, CD5, cyclin D1, SOX11, CD10, BCL6, MUM1, MYC, BCL2, and CD11c (see supplementary material, Table S2 for details of the antibodies used in this study). An Epstein–Barr virus‐encoded small RNA probe (DNP probe, Ventana, Tucson, AZ, USA) and an ISH iView Kit (Ventana) were used for Epstein–Barr virus detection. IHC and chromogenic in situ hybridization were performed using an automated immunostainer (BENCHMARK XT, Ventana).
At least one B‐cell marker (CD20, CD79a, or PAX5) with diffuse (almost 100%) expression and without T‐cell marker (CD3) expression was used to determine the B‐cell lineage. If at least two of the CD5, cyclin D1, and SOX11 biomarkers were positive (staining on >10% of the total tumor cells), pleomorphic mantle cell lymphoma was diagnosed and the patient was excluded from the cohort [14 (link)]. Hans algorithms were used to determine the cell‐of‐origin (COO) [1 ]; the thresholds for CD10, BCL6, and MUM1 were 30%, and the thresholds for MYC and BCL2 were 40 and 50%, respectively [1 ].