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The well-established cell-of-origin (COO) algorithm categorizes diffuse large B-cell lymphoma (DLBCL) into activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subgroups through gene expression profiling. We aimed to develop and validate a qPCR-based gene expression assay to determine the COO subgroups of DLBCL with formalin-fixed paraffin-embedded (FFPE) tissue. We first established a DLBCL transcriptome database of 1,016 samples retrieved from three published datasets (GSE10846, GSE22470, and GSE31312). With this database, we identified a qPCR-based 32-gene expression signature (DLBCL-COO assay) that is significantly associated with the COO subgroups. The DLBCL-COO assay was further validated in a cohort of 160 Chinese DLBCL patients. Biopsy samples from DLBCL patients with paired FFPE and fresh frozen tissue were collected to assign COO subtypes based on the immunohistochemistry (IHC) algorithm (Han's algorithm), DLBCL-COO assay, and global gene expression profiling with RNA-seq. For 111 paired FFPE and fresh DLBCL samples, the concordance between the IHC, qPCR, and RNA-seq methods was 77.5% and 91.9%, respectively. The DLBCL-COO assay demonstrated a significantly superior concordance of COO determination with the “gold standard” RNA-seq compared with the IHC assignment with Han's algorithm (P = 0.005). Furthermore, the overall survival of GCB patients defined by the DLBCL-COO assay was significantly superior to that of ABC patients (P = 0.023). This effect was not seen when the tumors were classified by the IHC algorithm. The DLBCL-COO assay provides flexibility and accuracy in DLBCL subtype characterization. These findings demonstrated that the DLBCL-COO assay might serve as a useful tool for guiding prognostic and therapeutic options for DLBCL patients.

Background: B cells can function as antigen-presenting cells by presenting antigens captured by the B-cell receptor (BCR) on Class II Major Histocompatibility Complex (MHC II) to T cells. In addition, B-cells can also maintain immune homeostasis by expressing PD-L1 and suppressing T-cell activity. Epstein-Barr virus (EBV) infection can disrupt B-cell function and lead to B cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Here we show that EBV-positive DLBCL (EBV+ DLBCL) has decreased expression of BCR and MHC II, but over-expressed PD-L1, which may lead to immune evasion. Methods: An EBV+ DLBCL cohort (n = 30) and an EBV- DLBCL control cohort (n = 83) were established. Immunostaining of PD-L1, MHC II, MHC II Transactivator (CIITA) and pBTK was performed on automated stainer. H-score was used to denote the results of staining of PD-L1 and pBTK. Break apart and deletion of CIITA locus was studied by fluorescent in situ hybridization. Surface immunoglobulin mean fluorescent insensitivity (MFI) was detected by flow cytometry to demonstrate the level BCR. Results: EBV+ DLBCL showed significantly lower expression of CIITA and MHC II compared to EBV- DLBCL. Genetic aberrations involving CIITA were also more common in EBV+ DLBCL, with 23% break apart events and 6% deletion events, comparted to 2% break apart and 0% deletion in EBV- DLBCL. In addition to the loss of antigen presentation molecule, the antigen capture receptor, BCR, was also down-regulated in EBV+ DLBCL. Accordingly, BCR signaling was also significantly decreased in EBV+ DLBCL as denoted by the respective pBTK levels. Conclusions: EBV+ DLBCL shows over expression of the T-cell inhibitory ligand, PD-L1. Antigen capture and presentation system were disrupted, and T-cell inhibitory molecule was hijacked in EBV+ DLBCL, which may contribute to immune escape in this high risk disease. Therapies targeting these aberrations may improve the outcome of patients with EBV+ DLBCL.