Angioimmunoblastic T-cell lymphoma (AITL) is one of the most common specific types of peripheral T-cell lymphomas (PTCL) 1. The cell of origin for AITL is T follicular helper cells (TFH) 2, 3. AITL has an immunophenotype closely akin to that of normal TFH; CD4+, CD8−, T-cell receptor (TCR) alpha/beta, and often expressing CXCL13, CD10, BCL6, Programmed-Death-1 (PD-1), ICOS, and CD200 2, 4–6. Diagnosis of AITL is challenging especially in small needle core biopsy due to the presence of admixed abundant inflammatory cells. The differential diagnosis is broad, and a definitive diagnosis requires a use of ancillary testing, including large panels of immunohistochemistry and molecular genetic studies. Immunophenotyping by flow cytometry is a common study for the diagnosis as well as for monitoring minimal residual disease of hematologic malignancies 7–13, but contribution of flow cytometry to diagnose AITL, while often reported, has been unclear due to lack of systemic studies with integrated morphologic assessment 14. Here, we evaluated the utility of highly sensitive flow cytometry in diagnosing AITL using an antibody against PD-1 in the context of other T-cell antigen. Tissue biopsies, bone marrow biopsies, and peripheral blood samples were retrieved from pathology archives between May 2015 and December 2018 at Memorial Sloan Kettering Cancer Center. This study was performed following the declaration of Helsinki and was approved by the institutional review board. The group was composed of 81 patients with PTCL and 40 patients with no T-cell lymphomas, but with potential morphologic mimics. Specifically, 36 patients with AITL, 6 with ALK-negative anaplastic large cell lymphoma, 2 with ALK-positive anaplastic large cell lymphoma, 9 with adult T-cell leukemia/lymphoma, 11 with peripheral T-cell lymphoma, not otherwise specified, 6 with nodal involvement by mycosis fungoides, 5 with T-cell large granular lymphocytic leukemia, 6 with T-cell prolymphocytic leukemia, 4 with nodular lymphocyte-predominant Hodgkin lymphoma, 1 with T-cell/histiocyte-rich large B-cell lymphoma, 5 with diffuse large B-cell lymphoma, germinal center B-cell type, 6 with follicular lymphoma, 12 with classical Hodgkin lymphoma, and 12 with benign reactive lymph nodes. Overall we obtained 94 tissue biopsies, 59 peripheral blood specimens, and 21 bone marrow aspirate specimens (Supplementary Table 1). Expression of PD-1 (CD279) on lymphoma cells was evaluated by BD FACSCanto 10-color flow cytometry (BD Biosciences, San Jose, CA) with BV605-conjugated anti-CD279 antibody (EH12. 2H7, BioLegend, San Diago, CA) along with other T-cell antigens frequently evaluated in this context. The results were analyzed with Woodlist software (Dr. BL Wood, University of Washington). Abnormal T-cell populations were identified by visual assessment of aberrant antigen expression. Mean fluorescence intensity (MFI) of anti-CD279 antibody was also evaluated. For selected cases, Beta Mark TCR Vbeta Repertoire Kit (Beckman-Coulter, Miami, FL) was used to confirm clonality. Flow sorting was performed in conjunction with molecular genetic analysis as a part of the study. Detailed methods are described in supplementary information. Flow cytometric analysis of all 12 reactive lymph nodes demonstrated that non-neoplastic T-cell population with

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