PD-L1 overexpression has also been described in cancer subtypes beyond those that have been so far considered for immune check point inhibitors [4, 10, 24C26], potentially opening this type of therapy to a larger and more diverse populations of cancer patients. further analyzed for gene copy number variations (CNV) by NGS and for co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently unfavorable (3/3). Co-amplification/CNVs of were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of alterations in cHL. Introduction Programmed cell death protein 1 (PD-1, encoded by gene) and one of its Mouse monoclonal to AURKA two known ligands, the programmed death ligand-1 (PD-L1, encoded by gene) are among the therapeutically most important checkpoint proteins that mediate tumor-induced WZ3146 immune suppression through T-cell downregulation . Their overexpression has been described in various solid tumors with marked clinical therapeutic effects due to the checkpoint blockade [anti-PD1/PD-L1 antibodies] , revolutionizing the treatment of solid malignancies, particularly metastatic melanoma, renal cell carcinoma, and non-small cell lung carcinoma (NSCLC). Patients with relapsed/refractory malignant lymphomas have limited therapeutic modalities and new WZ3146 therapeutic approaches are immensely important . Recent studies revealed the expression of PD-L1 among various B-cell lymphomas [4C6] with the most remarkable therapeutic benefits of PD-1 blockade in patients with Hodgkin lymphoma [3, 7]. PD-L1 status is usually determined by immunohistochemistry [8C10]. Food and Drug Administration (FDA) has recently approved PD-L1 22C3 antibody (DAKO pharmDx) as a companion diagnostics IHC kit for identifying non-small cell lung cancer (NSCLC) patients that are candidates for treatment with pembrolizumab. Several other antibodies (e.g. 28C8 clone from DAKO; SP142 clone and SP263 clone from Ventana) have been developed and used successfully in clinical trials for detection of PD-L1 protein expression in different tumor types (reviewed in ). Although PD-L1 overexpression is usually associated with greater clinical response (particularly to anti-PD1 antibodies) , the available clinical data indicate that only 10C30% tumors with PDL1 over expression respond to the PD-1/PD-L1 checkpoint inhibitors [11C14]. The reasons for this discrepancy might be due to different drugs, different antibody clones (validated for specific platforms, e.g. automated Ventana IHC systems or DAKO IHC autostainer), different thresholds, as well as complex pathophysiological mechanisms behind PD-L1 deregulation due to the interactions between cancer and immune cells [10, 15]. Several recent studies investigated the genetic basis of PD-L1 overexpression in tumors. In Hodgkin lymphoma alterations in chromosome 9p24.1 leads to (CD274) and (gene has also been described in triple-negative breast carcinomas [17, 18] and NSCLC [19, 20]. Green et al.  and Roemer et al.  also found gene alterations in classical Hodgkin lymphoma (cHL) while Georgiou et al.  recently demonstrated various cytogenetic alterations of gene in diffuse large B-cell lymphomas (DLBCL) including gains, amplifications and translocations. Genomic rearrangements of have also been described in primary mediastinal large B-cell lymphomas . A recent comprehensive survey of Budczies et al.  revealed frequent copy number variations (gains and amplifications [12%], deletions [31%]) across different cancer subtypes with direct impact on its protein and mRNA expression. In the present study, we explored the expression of PD-L1 in a diverse group of refractory/relapsed lymphomas. We compared the diagnostic power of two different anti-PD-L1 clones and also explored the genetic basis of PD-L1 overexpression analyzing gene along with and genes at 9p24 using in situ hybridization and next-generation sequencing (NGS) assays. Materials and Methods Samples and patients selection The study WZ3146 included 78 patients with refractory and/or resistant lymphomas of both B- and T-cell lineages. All lymphomas were diagnosed by a board-certified hematologist following the most recent lymphoma classification . A comprehensive immunohistochemical examination was WZ3146 performed for all those cases for the diagnostic purposes (e.g. CD30 for cHL, Fig 1B). Where appropriate, additional molecular assays (FISH, flow cytometry, PCR) were also performed. Epstein-Barr computer virus (EBV) status was available for 7 cases of which 5 cases were positive (2 cases of DLBCL of the brain and one case of lymphomatoid granulomatosis, classical Hodgkin lymphoma and peripheral T-cell.