Circulating Lymphoma Cells in Mycosis Fungoides/Sezary Syndrome Show Heterogeneity of the Cell-of-Origin and Variable PD-1 Expression By Flow Cytometric Analysis

Introduction: Mycosis fungoides (MF) is an epidermotropic primary cutaneous T-cell lymphoma. Its leukemic variant is recognized as Sezary syndrome (SS), although a study suggests that MF and SS may be distinct entities arising from different T-cell subsets; effector memory T-cells and central memory T-cells, respectively (Campbell JJ et al. Blood. 2010;116(5):767-771). Recent studies show bright PD-1 (T follicular helper (TFH)-like), and CD25/FOXP3 (Treg-like) expression in a subset of MF/SS cases by immunohistochemistry, and these findings raise the possibility that the cell-of-origin of MF/SS may be heterogeneous (Cetinozman F et al. Arch Dermatol. 2012;148(12):1379-1385, Prince HM et al. J Am Acad Dermatol. 2012;67(5):867-875, Satou A et al. Histopathology. 2016;68(7):1099-1108). In order to address this question comprehensively, we evaluated the expression levels of PD-1 on lymphoma cells of MF/SS patients by flow cytometry in peripheral blood (PB), and the results were compared to other T-cell lymphomas including angioimmunoblastic T-cell lymphoma (AITL). We also performed extensive flow cytometric immunophenotyping to algorithmically assess cell-of-origin of MF/SS with a subset of patients (Maecker HT et al. Nat Rev Immunol. 2012;12(3):191-200).

Methods: Patients who were diagnosed with T-cell lymphoma at Memorial Sloan Kettering Cancer Center between August 2015 and August 2018, and have circulating lymphoma cells in PB were selected for this study. Diagnosis of MF/SS was confirmed by skin biopsy along with the clinical presentation. PD-1 expression levels on lymphoma cells in PB were evaluated by 10-color flow cytometry including anti-CD279 (PD-1) antibody. Immunophenotyping to assess cell-of-origin of lymphoma cells (T-cell subset analysis) was performed with 3 tube/10-color flow cytometry, using the following antibodies: CD3, CD4, CD8, CD25, CD27, CD45RA, CD45RO, CD127, CD279, HLA-DR, CCR4, CCR6, CCR7, and CXCR3.

Results: Our study group is composed of 82 patients, including 34 MF/SS, 22 AITL, 2 anaplastic large cell lymphoma, ALK-negative (ALCL, ALK-), 8 adult T-cell leukemia/ lymphoma (ATLL), 5 peripheral T-cell lymphoma, NOS (PTCL-NOS), 5 T-cell large granular lymphocytic leukemia (T-LGL), and 6 T-cell prolymphocytic leukemia (T-PLL). The expression levels of PD-1 of lymphoma cells of the patients with MF/SS were widely variable (mean fluorescence intensity (MFI); Mean 949.0; range 101.0 - 3188.6) (Fig 1). 32.4% (11/34) of MF/SS cases showed high PD-1 expression equivalent to that of AITL cases. T-cell subset analysis to assess cell-of-origin was performed in 14 patients, including 4 patients with MF and 10 patients with SS (Table 1). While routine flow cytometric analysis showed similar immunophenotype other than the variation of PD-1, by flow cytometric T-cell subset analysis, we identified 3 patients with lymphoma cells showing T follicular helper (TFH) immunophenotype with CCR4+/CXCR3-/CCR6- and bright CD279 expression (case 2, 6, 8) (Table 2). Lymphoma cells of 8 patients showed effector memory CD4+ T-cell immunophenotype defined as CCR7-/CD45RA- (case 1, 3, 4, 5, 9, 10, 12, 13). Among these 8 patients, 2 patients had a subset of lymphoma cells showing central memory CD4+ T-cell immunophenotype defined as CCR7+/CD45RA- (case 5 and 12), and 2 patients had a subset with effector CD4+ cell immunophenotype defined as CCR7-/CD45RA+ (case 9, 13). 2 patients (case 7, 14) showed central memory CD4+ T-cell immunophenotype only, and 1 patient showed effector CD4+ T-cell immunophenotype only (case 11) (Table 2, Fig 2, 3). No cases showed Treg immunophenotype. The small sample size limited any subset analysis but we did not see obvious association between cell-of-origin of lymphoma cells and clinical features including prognosis, nodal presentation, number of circulating tumor cells, and histological findings in this study with limited number of cases.

Conclusions: Lymphoma cells in MF/SS show marked heterogeneity of expression of PD-1 including clear subset arising from TFH. This suggests MF/SS may represent multiple biological entities. Further studies will be necessary to investigate the clinical significance of cell-of-origin of MF/SS.

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