The Role of Tumor Microenvironment in the Pathogenesis of Sézary Syndrome

Sézary syndrome is an aggressive leukemic variant of cutaneous T-cell lymphomas, characterized by erythroderma, lymphadenopathy, and peripheral blood involvement by CD4+ malignant T-cells. The pathogenesis of Sézary syndrome is not fully understood. However, the course of the disease is strongly influenced by the tumor microenvironment, which is altered by a combination of cytokines, chemokines, and growth factors. The crosstalk between malignant and reactive cells affects the immunologic response against tumor cells causing immune dysregulation. This review focuses on the interaction of malignant Sézary cells and the tumor microenvironment.

Sézary syndrome preceded by mycosis fungoides and complicated by tumor lysis syndrome and macrophage activation syndrome

Sir, Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common malignancies of cutaneous T-cell lymphoma [1]. Herein, we report a case of SS complicated by tumor lysis syndrome and macrophage activation syndrome. A 54-year-old patient, followed since October 2017 for mycosis fungoides and undergoing various treatments (PUVA therapy, methotrexate, chlorambucil + prednisone), presented with an aggravation of lesions toward extensive and intensely pruritic. A clinical examination revealed dry erythroderma, scratch marks, wart plaques, an accentuation of frontal wrinkles and nasolabial folds (leonine facies), palmoplantar fissuring keratoderma, xanthopachyonychia of all nails, and a carapace-like appearance of the scalp (Figs. 1 – 3). Generalized lymphadenopathy, hepatomegaly, and a state of anasarca-type edema caused by hypoalbuminemia were also found. A skin biopsy revealed lymphoproliferation of CD4+ T-cells and an aberrant loss of pan-T antigens. The CD4-to-CD8 ratio was at 48.5% and Sézary cells were 6960 (absolute value). A lymph node biopsy showed a dense infiltration of Sézary cells. A PET scan revealed hypermetabolism in the entire skin and at the lymph node level. Tumor lysis syndrome was evident, with high levels of blood uric acid (at 182 mg/L), elevated LDH (at 924 U/L), and functional kidney failure. Macrophage activation syndrome was also present, with fever, anemia and thrombocytopenia, liver cytolysis, hypertriglyceridemia, and hyperferritinemia. The patient received an albumin infusion, oral corticosteroid therapy to treat the syndrome, and rasburicase for hyperuremia. Despite this, the patient died before multiagent chemotherapy could have been started. On rare occasions, SS may be preceded by a prior history of classic MF. The International Society for Cutaneous Lymphomas (ISCL) recommends that such cases be designated “SS preceded by MF” [2]. Traditionally, SS is defined as a leukemic form of CTCL associated with erythroderma. Sézary cells are a population of large lymphocytes in the peripheral blood, with grooved and lobulated nuclei, in the case of SS, numbering 1000 cells/mL or more [2]. The histopathologic findings in the skin often resemble those observed in MF, with less prominent epidermotropism. As in MF, immunohistochemical studies showing a CD4 predominance and loss of pan-T-cell markers may be helpful. Lymph node involvement is characterized by the complete effacement of the nodal architecture by the infiltrating Sézary cells (2). The poor prognostic factors in Sézary syndrome include an advanced stage of the disease, an older age at onset, and large cell transformation [3]. While high response rates may be achieved with systemic chemotherapy, they are frequently short-lived and associated with significant toxicities [2]. The management of SS is complicated and requires multidisciplinary collaboration between dermatologists, hematologists, biologists, and reanimators.

ICOS Is Widely Expressed in Cutaneous T-Cell Lymphoma and Its Targeting Promotes Potent Killing of Malignant Cells

Background: Advanced cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), do not deliver significant long-term improvements in patient outcomes. More recently, mogamulizumab and anti-KIR3DL2 provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest. Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. Skin samples from 12 patients with B-cell lymphomas, 14 with CD30 + lymphoproliferative disease (LPD), 12 with primary cutaneous CD4+ small/medium T-cell lympho-proliferative disorder and 13 with angioimmunoblastic T-cell lymphoma (AITL) were used as controls. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes. Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS 314.8 monoclonal antibodies with MMAE and pyrrolobenzodiazepine (PBD), in comparison to BV. We used ICOS + CTCL cell lines (MyLa, MJ and HUT78), murine xenograft models with MyLa and ICOS + Patient Derived Xenografts (PDXs) from patients with SS and AITL. In order to identify the best anti-ICOS clone that we should develop for a clinical trial, we evaluated the affinity of the antibody on the receptor, the internalization capacity of the antibody using pHAb Reactive Dyes kit (Promega®), and the ability of the antibody to act as an ADC using a secondary conjugate (Mab-ZAP kit, Advanced Targeting Systems®). Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in all the involved nodes. Double staining experiments which were performed in both skin and lymph node revealed that ICOS expression appears mainly restricted to neoplastic CD4 + T-cells, with rare ICOS +CD8 + T-cells in the tumor micro-environment. ICOS expression by circulating Sézary cells was strong: 69 ± 7.3% versus 38.8 ± 7.1% of non-tumoral CD4 + cells (p<0.009; CI95%: 8.7-51.6); and 31 ± 3.2% of CD4+ cells in HD (p<0.0001; CI95%: 20.3-46.3). Percentages of ICOS + Tregs were significantly higher in patients with SS than in HD. In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS-MMAE (IC50 = 8.2ng/mL) and anti-ICOS-PBD (IC50 = 1.2ng/mL) ADCs. In a mouse xenograft model (MyLa), anti-ICOS-MMAE ADCs provided a longer overall survival (OS) than BV (HR=15.2; CI95%: 3.2-71.1; p<0.0006). Finally, in ICOS + PDXs, anti-ICOS-MMAE ADCs significantly improved OS, and reduced the number of tumor cells in the blood, spleen, and bone marrow. No evidence of ADC toxicity was observed in treated mice. Among 8 different anti-ICOS clones, clone 314.8 had the best affinity on MyLa and MJ cell lines. Clones 53.3, 293.1, 92.17, 88.2 and 279.1 also had good affinity to receptor, whereas clones 145.1 and 121.4 had poor affinity. Using the internalization pHAb reactive dyes kit, we found that clones 314.8, 53.3, 92.17, 88.2 internalized significantly better and faster than the other ones. In order to verify if there is a correlation between internalization capacity and ADC effect, clones 53.3, 92.17 and 145.1 were coupled to MMAE. While anti-ICOS-53.3 and anti-ICOS-92.17 ADCs had similar efficacy to anti-ICOS-314.8 ADCs on MyLa, anti-ICOS 145.1 ADCs resulted in significantly lower cell death. Finally, all clones showed good ability to act as ADCs with Mab-ZAP, excluding clones 279.1, 145.1 and 121.4. Discussion: ICOS is a promising therapeutic target because it is expressed both by tumor T-cells and regulatory T-cells. We report for the first time the strong and frequent expression of ICOS in CTCLs, as well as the preclinical efficacy of anti-ICOS ADCs on CTCL cell line and PDXs. These results could be extended to the spectrum of follicular variant peripheral T-cell lymphomas. Conclusion: Collectively, our findings provide the preliminary basis for a therapeutic trial Figure 1 Figure 1. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Chimerized Anti-ICOS 314.8 Monoclonal Antibodies Inhibit Tumor Cells and Regulatory T Cells in Patients with Sézary Syndrome

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy. Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®). Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry. Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS. Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered. ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1). Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031). Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p<0.01; CI95%:-53--27). The efficacy was better than with alemtuzumab, but there was no significant difference with moga. The ADCP effect induced by anti-ICOS did not differ with moga. Interestingly, anti-ICOS were effective in 6 moga-resistant patient, as they induced 38.9±5.9% of apoptosis, compared to 12.4±4.7% with moga (p<0.001). Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab. Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs’ pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

CD158k and PD-1 expressions define heterogeneous subtypes of Sezary syndrome

Sezary syndrome (SS) is a rare leukemic form of cutaneous T-cell lymphoma. Diagnosis mainly depends on flow cytometry, but results are not specific enough to be unequivocal. The difficulty in defining a single marker that could characterize Sezary cells may be the consequence of different pathological subtypes. In this study, we used multivariate flow cytometry analyses. We chose to investigate the expression of classical CD3, CD4, CD7, and CD26 and the 2 new association of 2 markers CD158k and PD-1. We performed lymphocyte computational phenotypic analyses during diagnosis and follow-up of SS patients to define new SS classes and improve the sensitivity of the diagnosis and the follow-up flow cytometry method. Three classes of SS, defined by different immunophenotypic profiles, CD158k-positive SS, CD158k-negative PD-1-positive SS, CD158k and PD-1 double-negative SS, showed different CD8+ and B-cells environments. Such a study could help to diagnose and define biological markers of susceptibility/resistance to treatment including immunotherapy. -

Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome

Sézary syndrome (SS) is an aggressive leukemic form of Cutaneous T-cell Lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 SS patients employing standardized and sensitive EuroFlow-based multiparameter flow cytometry (MFC). We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter-and intra-patient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and non-classical T helper subsets with different maturation phenotypes.

Sézary Syndrome with CD4/CD8 Double-Negative Neoplastic T Cells in Peripheral Blood

Sézary syndrome is a rare leukemic type of cutaneous T-cell lymphoma characterized by the presence of neoplastic T cells with cerebriform nuclei (Sézary cells) in the skin, lymph nodes, and peripheral blood. Typical Sézary cells have a CD3+CD4+CD8– phenotype; however, in cases of the aberrant loss of antigens on Sézary cells, especially the loss of critically important T-cell antigens such as CD4, there is a possibility of misdiagnosing the disease or underestimating the tumor burden of the disease. Here, we report a rare case of Sézary syndrome with CD4/CD8 double-negative Sézary cells in the peripheral blood. Most of the Sézary cells in the peripheral blood had lost CD4 expression, and we diagnosed the disease and evaluated the tumor burden by multicolor flow cytometry. Intriguingly, the Sézary cells showed a typical CD4+CD8–CD7– phenotype in the skin even though the cells in the peripheral blood lacked CD4. The patient responded well to treatment with bexarotene and narrow-band ultraviolet B therapy. Analysis by multicolor flow cytometry is essential to diagnose this rare type of Sézary syndrome and evaluate the tumor burden.