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 (TN), central memory (TCM), effector memory (TEM), or effector memory with reacquired CD45RA (TEMRA); 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.

INTRODUCTION

Mycosis fungoides (MF) and Sézary syndrome (SS) are two clinically distinct cutaneous T-cell lymphomas (CTCLs) that share marked morphologic and phenotypic similarities, despite distinctively different clinical presentations. MF is the most common CTCL, with an incidence of 0.36–0.55 per 100,000 individuals in the United States (1,2), and characterized clinically by slowly progressive patch, plaque and tumor lesions (3). A subset of patients with MF progresses to more aggressive phase of the disease with large cell transformation or leukemic MF. In contrast, SS is a rare CTCL with an incidence of 0.01 per 100,000 individuals in the United States (2), and presents de-novo with diffuse erythroderma, lymphadenopathy, and overt leukemic disease. Both SS and MF cells are Sézary cells with cerebriform nuclei and are CD4⁺/CD8⁻ αβ T-cells in most cases. Both exhibit similar T-cell antigen expression profiles when studied on peripheral blood, with frequent loss of CD26 and CD7 (4). Patients with MF may also develop diffuse erythroderma during the course of the disease (5). These cases of erythrodermic MF can have lymphadenopathy and circulating Sézary cells in the peripheral blood, and thus are indistinguishable clinically from SS. Nevertheless, patients with erythrodermic MF usually have a history of MF with patch/plaque lesions for years and should not be diagnosed as SS, which presents with erythroderma and peripheral blood involvement at initial presentation.

Advances in our understanding of T-cell immunology have stimulated an interest in determining the putative cell of origin of various T cell lymphomas, including CTCLs, based on their immunophenotypic resemblance to known benign T-cell subsets. Various discrete subpopulations of normal CD4⁺ T-cells have been described, corresponding to different stages of antigen experience, functional properties, migration capabilities and their ability to survive and/or self-renew (6–9). T-cells emigrating from the thymus that have not yet encountered their cognate antigen constitute the naïve T-cell subset (T_N), which has the capacity to self-renew and home to secondary lymphoid tissues. When T-cells encounter their cognate antigen, they progress through central memory T-cells (T_CM), effector memory T-cells (T_EM), and finally terminally differentiated effector memory T-cells (T_EMRA). T_CM cells retain the capacity to home to secondary lymphoid tissues and self-renew, providing the basis for long term immunity. T_EM cells are capable of migrating to sites of inflammation and best suited to exert their function as orchestrators of the immune response. T_EMRA cells develop cytotoxic properties and express IFN-γ after activation through the T-cell receptor (TCR). The CD8⁺ T_EMRA cells have been well characterized, but the CD4⁺ T_EMRA cells are ill-defined and usually ignored in the literature, although both CD4⁺ and CD8⁺ T_EMRA cells have similar functions. The progression of T-cells from T_N to T_EMRA cells is accompanied by characteristic changes in antigen expression of surface markers including CD45RA, CD45RO, CCR7, CD62L, CD27, and CD28, which can be used to define the T-cell subsets. T_N cells are CD45RA⁺CD45RO⁻CCR7⁺CD62L⁺CD27⁺CD28⁺. After antigen stimulation, T_N cells lose CD45RA and gain expression of CD45RO and become T_CM cells. As T_CM cells progress to T_EM cells, they further lose expression of CCR7, CD62L, CD27, and CD28. At the last stage, T_EMRA cells regain expression of CD45RA with loss of CD45RO. T_EMRA cells are otherwise differentiated from T_N cells by the absence of expression of CCR7, CD62L, CD27, and CD28.

Immunophenotypic analyses on a limited number of CTCL cases have suggested that MF originates from T_EM, while SS arises from T_CM, indicating that MF and SS are biologically distinct diseases (10–12). This concept has been widely embraced, as it helps explain the dramatically different clinical behavior of the two diseases with otherwise indistinguishable cytologic and immunophenotypic features (13,14). Nevertheless, recent studies have found a more heterogeneous expression (15–17), challenging the above cell-of-origin hypothesis.

Using a 10-color flow cytometry T-cell panel, we prospectively analyzed the immunophenotypes of neoplastic T-cells in clinically defined MF and SS, detected on fresh peripheral blood or bone marrow samples. Our findings reveal a broad heterogeneity and variability in the expression of antigens associated with naïve or memory T-cell subsets, with no significant correlation with disease classification based on clinical features. Moreover, significant phenotypic shifts on the expression of these antigens were detected on follow-up specimens, suggesting that the naïve/memory phenotype of the neoplastic cells is likely representative of a functional state, rather than an indication of cell of origin.

MATERIALS AND METHODS

Patient Selection

Patients with MF or SS were evaluated at Moffitt Cancer Center (MCC) between February 2015 and October 2017. Staging studies performed upon initial evaluation included complete physical examination, computer tomography, peripheral blood cell counts, white blood cell differentials, and peripheral blood flow cytometry, following the 2007 criteria from the International Society for Cutaneous Lymphoma (ISCL) and the European Organization of Research and Treatment of Cancer (4). Histological slides, flow cytometry results, TCR gene rearrangement studies, imaging studies, photographs of skin lesions, and clinical history were evaluated at the MCC interdisciplinary tumor board to render a clinicopathologic diagnosis based on the World Health Organization/European Organization for Research and Treatment of Cancer (WHO/EORTC) cutaneous lymphoma guidelines (18), and recommendations from the ISCL (19). Control peripheral blood samples were retrospectively identified by searching the medical records for patients with a complete clinical history, comprehensive laboratory work up and no demonstrable hematological malignancy, on which a 10-color T-cell panel was performed. This study was approved by the University of South Florida Institutional Review Board.

Flow Cytometry

All samples were analyzed fresh and stained with antibodies within 24 hours after collection. Aliquots of 50 μL of whole blood or bone marrow aspirate in sodium heparin, were incubated in the dark for 15 min at room temperature with combinations of 10 monoclonal antibodies conjugated to Pacific Blue (PB), Krome Orange (KO), fluorescein isothiocyanate (FITC), phycoerythrin (PE), R-phycoerythin-Texas Red-X (ECD), phycoerythrin-cyanine 5.5 (PC5.5), phycoerythrin-cyanin 7 (PC7), allophycocyanin (APC), allophycocyanin-alexa fluor 700 (APC-A700), and allophycocyanin-alexa fluor 750 (APC-A750). Red blood cells were lysed with BD FACS lysing solution (BD Biosciences, San Jose, CA) and nucleated cell were resuspended in phosphate buffered saline containing 2% paraformaldehyde. A standardized 10-color T-cell panel (PB/KO/FITC/PE/ECD/PC5.5/PC7/APC/APC-A700/APC-A750) was utilized in all specimens, consisting of three stained tubes (CD8/CD45/CD2/CD26/CD3/CD5/CD7/CD30/CD19/CD4, CD8/CD45/TCRγδ/TCRαβ/CD3/PD-1/CD10/CCR4/CD25/CD4, and CD57/CD45/CD45RA/CD52/CD62L/CD56/CD7/CD16/CD4/CD3). All antibodies were obtained from Beckman Coulter (Brea, CA). Up to 1,000,000 events were acquired until 5,000 lymphocytes were collected on a Gallios flow cytometer (Beckman Coulter).

Listmode files were analyzed as previously described (20), based on ad-hoc templates created on Kaluza version 1.5a (Beckman Coulter, Brea, CA). In brief, each lymphoid population was defined as the intersection of three interdependent polygonal gates drawn on three separate two-dimensional dot-plots, representing the six most informative and biologically relevant parameters for each particular lymphoid subset. Real time color-coding for each population was used to visually narrow the polygonal gates in order to define single clusters of cells with homogeneous fluorescence properties in most parameters studied. All lymphoid populations were identified and categorized.

Delta median fluorescence values were calculated by comparing the fluorescence of gated neoplastic events to that of monocytes (for CD2, CD5, CD7, CCR4, and PD-1), neutrophils (for CD26 and CD52) or NK cells (for CD3 and CD4) within the same analysis tube.

Statistical Analysis

All statistic calculations were performed using GraphPad Prism, version 7.01 for Windows (GraphPad Software, San Diego, CA). Differences in antigen expression between groups were studied using a two-tailed Mann Whitney test on delta median fluorescence values. Differences in frequencies of antigen loss or naïve-memory T-cell phenotype were studied using the Chi-square test. A statistically significant P value was considered as <0.05.

RESULTS

We identified neoplastic T-cells by 10-color flow cytometry on 74 peripheral blood specimens and 5 bone marrow specimens from 52 patients with CTCL. Based on a comprehensive review of the clinical history, laboratory studies, and pathology material, 33 patients met criteria for de-novo SS while 19 patients were diagnosed as MF. The median age of the patients with SS was 71 years old (range: 28–90), and the male to female ratio was 1.4. At the time of diagnosis, prior and current staging studies were consistent with stage IVA1 (27 patients, 82%), IVA2 (2 patients, 6%), or IVB (4 patients, 12%) disease in patients with SS. The median age of the patients with MF was 69.5 years old (range: 33–87), and the male to female ratio was 5.7. At the time of initial diagnosis of MF, patients presented with stage IA (3 patients, 16%), IB (9 patients, 47%), IIB (3 patients, 16%), IIIA (1 patient, 5%), IIIB (1 patient, 5%), and IVA1 (2 patients, 11%) disease.

To standardize our immunophenotypic analysis, we studied 27 control individuals (patients with no demonstrable hematologic malignancy) and showed the presence of discrete CD4⁺ T-cell subsets corresponding to T_N (CD62L⁺/CD45RA⁺, median: 21.8%, range: 2.2–74.3%), T_CM (CD62L⁺/CD45RA⁻, median: 51%, range: 19.9–70%), T_EM (CD62L⁻/CD45RA⁻, median: 21.3%, range: 4.17–59.4%), and T_EMRA (CD62L⁻/CD45RA⁺, median: 1.2%, range: 0–11%, Figure 1A,B). In contrast, CD8⁺ T-cells showed a more prominent population of T_EMRA cells (range: 1–72.8%, median: 29.6%) (Figure 1C), while most NK cells were positive for CD45RA and showed a spectrum or biphasic expression of CD62L (Figure 1D). Based on these findings, appropriate thresholds were set up on CD62L versus CD45RA 2-dimensional plots for the evaluation of tumor samples, with particular attention to background benign lymphoid subsets as internal controls (Figure 2).

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.

Immunophenotypic results for all cases are summarized in Figure 3. With the exception of 1 case of MF double negative for CD4/CD8, all neoplastic cells were positive for CD4, and negative for CD8 and TCRγδ. Except for one patient (four cases) with neoplastic cells lacking surface TCR expression (as determined by absent surface CD3 and TCR), all cases were positive for TCRαβ. As previously described (20), the most common immunophenotypic aberrancies in SS and MF were decreased expression or loss of CD26 (81%) and CD7 (64.6%), followed by variably diminished expression of CD2 (41.8%), CD3 (35.4%), CD4 (30.4%), and CD5 (15.2%). There was no statistically significant correlation between the incidence of these immunophenotypic abnormalities and a clinical diagnosis of MF or SS (data not shown). When gated based on these immunophenotypic aberrancies, neoplastic T-cells comprised a median of 58.8% of lymphoid events (range: 10–97.4%).

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).

Gated neoplastic T-cells from patients with CTCL exhibited a variety of staining patterns for CD62L and CD45RA (Figure 2). When comparing cases of MF and SS, the predominant naïve/memory phenotype was T_N in 12% and 15% of cases, T_CM in 33% and 46% of cases, T_EM in 49% and 30% of cases, and T_EMRA in 6 and 9% of cases, respectively (Figures 3). There was no correlation between naïve/memory phenotype and a clinical diagnosis of MF versus SS (P = 0.4). When sequential samples from the same patient were compared, the naïve/memory phenotype remained the same for 10 patients, up to 273 days after the initial evaluation. In six other patients, the naïve/memory phenotype was different on one or more follow up analyses (Figure 4), due to changes in CD62L expression (switching between T_CM and T_EM phenotype) in five cases, and loss of CD45RA expression in one case.

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).

Expression of the targetable surface antigens CD52, CCR4, and PD-1 was evaluated on CTCL patients and controls, excluding samples obtained after therapy with the corresponding therapeutic antibody (Alemtuzumab, Mogamulizumab, and anti-PD1 antibodies, respectively). As a group, neoplastic T-cells from patients with CTCL exhibited higher expression of PD-1 and CCR4 (P < 0.0001 for both), compared to benign CD4 T-cells from control individuals; although expression levels comparable with benign T-cells were observed in many cases (Figure 5). With the exception of 3 CTCL cases from different patients negative for CD52 (4 SS and 1 MF), neoplastic cells were positive for CD52 at levels comparable to benign T-cells (P = 0.36). SS cases exhibited higher levels of CCR4 compared to MF cases (P = 0.001), but with significant overlap of fluorescence values. In addition, negative PD-1 expression was not seen in SS but encountered in a few cases of MF (Figure 5B).

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.

DISCUSSION

Prior immunophenotypic studies on limited numbers of CTCL cases have reported that neoplastic T-cells in SS are usually positive for CD27, CCR7, and CD62L; while neoplastic T-cells from skin lesions in MF are usually negative for these surface antigens (10,11). In the normal CD4⁺ T-cell repertoire, expression of these antigens is strongly associated with the T_N and T_CM subsets, while lack of expression is characteristic of the T_EM and T_EMRA subsets. CD62L and CCR7 play an essential role in T-cell migration to lymph nodes while CD27 mediates costimulatory signals necessary for the establishment of T-cell immunity, consistent with the physiologic role of T_N and T_CM as circulating immune initiators and surveyors. In addition, CTCL has been shown to express surface molecules involved in T-cell homing to skin (namely CLA, CCR4, and CCR10), which can also be demonstrated in a subset of antigen-experienced benign T-cells. This evidence has fueled the concept that SS originates from CD4⁺ T_CM cells, while MF arises from T_EM cells.

The above “cell-of-origin” hypothesis has gained widespread support, as it helps explain the dramatic differences in clinical behavior between MF and SS, despite otherwise almost identical cytological and immunophenotypic features. Nevertheless, other lines of evidence have posed a challenge to this model. For example, at least two reports have demonstrated a high incidence of CCR7 expression in MF, particularly in advance-stage skin lesions (16,17), arguing against a T_EM phenotype. When studied, CD45RA expression in MF and SS has been variable and not supportive of a T_CM or T_EM phenotype, as this marker is expected to be negative in these T-cell subsets (21). More recently, two immunophenotypic studies have revealed an unexpected heterogeneity of the naïve/memory maturation phenotype in SS, with remarkable disparities between blood- and skin-derived neoplastic T-cells (22,23). In addition, mass parallel gene sequencing has demonstrated a high number of ultraviolet light-associated dinucleotide mutations (CC> > TT) in both MF and SS (24,25), arguing against a T_CM origin as this T-cell subset is largely circulatory and not skin-bound. We hereby report the largest published series of CTCL evaluated for naïve/memory phenotypic properties on neoplastic T-cells by flow cytometry. Importantly and for the first time, we compared the naïve/memory phenotype of circulating neoplastic T-cells in SS and MF on a large clinical series, to evaluate the veracity of the “cell of origin” hypothesis and the diagnostic utility of naïve/memory phenotyping to distinguish SS from leukemic MF. As suggested by recent lines of evidence, we demonstrated a highly variable spectrum of naïve/memory T-cell phenotype in CTCL, with no statistically significant correlation with a clinical diagnosis of MF versus SS.

Our naïve/memory T-cell phenotyping is based on two surface antigens widely utilized for this purpose: CD62L and CD45RA. It should be noted that the correlation between these and other markers utilized to define T-cell subsets, such as CD27, CD44, CD45RO, CD127, and CCR7, is not perfect when studying the normal CD4⁺ T-cell repertoire (8,21) and also on limited data on CTCL (10). Thus, attempts to identify naïve or memory-like phenotypes in CTCL using a different set of antigens might not give identical results. In the design of our panel, we favored using CD62L over the similarly expressed antigen CCR7, based on the essential role that CD62L plays in the rolling of lymphocytes on high endothelial venules, and the fact that CXCR4 (which is commonly expressed in CTCL) can compensate for a lack of CCR7 in facilitating T-cell homing to lymph nodes (26). Similarly, CD45RA expression was arbitrarily selected (over lack of CD44 and CD45RO expression) as the marker of choice to define T_N cells, based on our desire to also identify the T_EMRA subset.

CD62L (also known as l-selectin) is a cell adhesion molecule, which recognizes sialylated carbohydrate groups on CD34, GlyCAM, and MadCAM expressed on high endothelial venules. It is an indispensable surface molecule for the rolling and tethering of leukocytes entering lymphoid tissues, and it is believed to mediate the circulation of the T_N and T_CM subsets through lymph nodes. Interestingly, CD62L-positive neoplastic cells have been shown to be selectively depleted upon anti-CD52 (Alemtuzumab) therapy in CTCL (12), suggesting a mechanism for the resistance of CD62L-negative skin infiltrates in MF. In our series, we demonstrate that the expression of CD62L in circulating Sézary cells is highly variable, both in MF and SS, and is not necessarily stable. These findings might have important implications for therapy with Alemtuzumab and should be further studied in the setting of clinical trials.

The phenotypic shift observed in 37.5% of the patients (6 of 16 patients) was mainly due to changes in CD62L expression. These patients were not treated with Alemtuzumab. While CD62L expression is often lost after density gradient centrifugation and cryopreservation of samples, the change of CD62L was not believed to be due to these technical issues as all our samples were analyzed fresh and comparably between initial and subsequent samples. MF has been postulated to result from persistent antigen stimulation, and the change in the naïve/memory phenotype is an antigen driven event (27). Therefore, the phenotypic shift in these patients was most likely driven by an unknown antigenic challenge. This further supports the idea that the naïve/memory phenotype of CTCL reflects the functional status of the Sézary cells and is not a good surrogate for the classification of SS and MF.

CCR4 is the chemokine receptor for CCL17 and CCL22, and has been involved in the migration of activated T-cells into inflamed tissues, including skin. Indeed, CCR4 has been shown to mediate the migration of tumor cells from blood to skin in SS (28). Approximately 20–25% of peripheral blood CD4⁺ T-cells from healthy individuals exhibit some level of expression of CCR4 (29,30), while CCR4 expression in MF and SS has been reported to be consistently positive (10–12) and higher than benign T-cells (30). In one study, higher levels of CCR4 expression were observed in SS compared to MF, which correlated with a better response to therapy with the anti-CCR4 antibody Mogamulizumab (31). We have independently confirmed a statistically significant higher expression of CCR4 in SS compared to MF, in agreement with the above reported finding. Although assessment of CCR4 expression by flow cytometry provides useful information when considering therapy with Mogamulizumab, significant overlaps of CCR4 expression between MF, SS, and reactive T-cells might pose challenges in defining and harmonizing positivity thresholds and expression intensity measures.

In conclusion, we report the immunophenotype of circulating MF and SS, defined based on expert multidisciplinary review of the clinical history and laboratory studies. In particular, we defined leukemic/advanced-stage MF based on a reliable history depicting clinical features typical for MF and eventual progression to leukemic disease. While MF and SS are recognized as two distinct CTCLs in the current WHO Classification of Diseases (32), ours, and other recent findings indicate that circulating MF and SS show similar naïve/memory maturation phenotypic heterogeneity and may represent a spectrum of same disease process. Thus other than the clinical history, the cases of “MF transformation to SS” or “secondary SS” cannot be reliably distinguished from “De novo” SS based on phenotypic analysis.