Naïve/memory T-cell phenotypes in leukemic cutaneous T-cell lymphoma: Putative cell of origin overlaps disease classification

Abstract

Background: Mycosis fungoides (MF) and Sézary Syndrome (SS) are clinically distinct cutaneous T-cell lymphomas with strikingly similar morphologic and phenotypic features. Prior studies have suggested phenotypic differences based on markers of antigen experience, suggesting a different cell of origin.

Methods: Seventy-nine involved peripheral blood or bone marrow samples from 33 patients with SS and 19 patients with MF were studied by 10-color flow cytometry, including CD62L, CD45RA, CCR4, and PD-1. Gated tumor events were classified as naïve (T_N ), central memory (T_CM ), effector memory (T_EM ), or effector memory with reacquired CD45RA (T_EMRA ); based on CD62L⁺ /CD45RA⁺ , CD62L⁺ /CD45RA^- , CD62L^- /CD45RA^- , or CD62L^- /CD45RA⁺ phenotype, respectively. Sequential specimens were compared to assess for phenotypic stability.

Results: The naïve/memory phenotype of the neoplastic T-cells was markedly heterogeneous, with a dominant T_N , T_CM , T_EM , or T_EMRA subset on 11 (14%), 32 (41%), 30 (38%), and 6 (8%) cases, respectively. There was no correlation between the diagnosis of MF or SS and putative cell of origin (P = 0.4). Overexpression of CCR4 and PD1 was observed in most cases, with higher intensity in SS compared to MF. The naïve/memory phenotype remained the same for 10 patients up to 273 days after the initial analysis; while on six patients, the naïve/memory phenotype was different from the original phenotype.

Conclusions: Both SS and MF can have phenotypic features of any of the major naïve/memory T-cell subsets, which questions the current principle of "cell-of-origin" distinction between SS and MF. Phenotypic shifts within these subsets are common, suggesting a functional state rather than a cell-of-origin surrogate. © 2018 International Clinical Cytometry Society.

Keywords: Sézary syndrome; flow cytometry; memory T-cells; mycosis fungoides; naïve T-cells.

Fig. 1.

A, Representative flow cytometry plots from a control individual with no demonstrable hematologic malignancy, showing the typical distribution of benign CD4⁺ T-cells (cyan), CD8⁺ T-cells (orange), and NK cells (gold), on CD62L versus CD45RA dot plots. Also shown are the quadrant gates utilized to quantify naïve (T_N), central memory (T_CM), effector memory (T_EM), and effector memory with CD45RA expression (T_EMRA) CD4⁺ T-cells. B, Percentages of T_N, T_CM, T_EM, and T_EMRA CD4⁺ T-cells, and C, similar subsets of CD8+ T-cells across 27 control individuals. D, Percentage of NK cells falling within the quadrant gates defining naïve/memory T-cell subsets.

Fig. 2.

Representative peripheral blood flow cytometry plots from patients with MF (A and C) or SS (B and D), demonstrating the presence of neoplastic T-cells with immunophenotypic features of T_N (A), T_CM (B), T_EM (C), or T_EMRA cells (D). Neoplastic T-cells (red) were identified based on immunophenotypic aberrancies, such as CD3^dim/CD4^dim (A), CD3^dim/CD26⁻ (B), CD2^dim/−/CD26⁻ (C), and CD3⁺/CD2⁻ (D). Background benign CD4⁺ T-cells (cyan), CD8⁺ T-cells (orange), and NK cells (gold) are also shown. Expression of PD-1 and CCR4 is shown in the middle plots.

Fig. 3.

Heat maps summarizing the flow cytometric findings in leukemic CTCL, including the presence of neoplastic T-cells with predominantly T_N, T_CM, T_EM, or T_EMRA phenotype. Each row represents gated benign CD4⁺ T-cells from individuals with no hematopoietic malignancy (top), or gated aberrant neoplastic T-cells from patients with MF or SS. The color intensity on the left heat maps depicts the percentage of benign CD4⁺ T-cells (top) or neoplastic T-cells within the corresponding naïve/memory T-cell subset-defining quadrants, on a CD62L versus CD45RA dot plot. The color intensity on the right heat maps depicts the delta median fluorescence intensity (ΔMFI) compared to an internal control population, and normalized to the highest recorded value for the corresponding antigen (% maximum value).

Fig. 4.

Representative peripheral blood flow cytometry plots from two patients with Sezary syndrome exhibiting naïve/memory phenotype shifts on follow up studies. A, CD3^dim/CD26⁻ neoplastic T-cells showed a similar T_CM phenotype at diagnosis (not shown) and 27 days later (top). On day 234, a follow up study showed loss of CD62L expression on the neoplastic cells, corresponding to a T_EM phenotype (bottom). B, CD3⁺/CD2^dim/− neoplastic T-cells at first evaluation showed a T_EM phenotype (top). On a follow up study 277 days later, the neoplastic cells showed gain of CD62L expression and a T_CM phenotype (bottom).

Fig. 5.

Scatter plots comparing the level of expression of CD52 (A), PD1 (B), and CCR4 (C) between benign CD4⁺ T-cells from control individuals, and malignant T-cells identified on samples from patients with MF and SS. Each dot represents one sample studied. For illustration purposes, values ≤0.1 is displayed against the axis. ΔMFI: Delta median fluorescence intensity, compared to an internal control population. Error lines show median and interquartile range. The P values for statistically significant differences are shown. NS: Not statistically significant.