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.

High IKZF2 Expression in Malignant T cells Promotes Disease Progression in Cutaneous T cell Lymphoma

Cutaneous T cell lymphoma is a generally indolent disease derived from skin-homing mature T cells. However, in advanced stages, CTCL may manifest as aggressive clinical behavior and lead to a poor prognosis. The mechanism of disease progression in CTCL remains unknown. Here, with a large clinical cohort, we identified that IKZF2, an essential transcription factor during T cell development and differentiation, showed stage-dependent overexpression in the malignant T cells in MF lesions. IKZF2 is specifically over-expressed in advanced-stage MF lesions, correlates with poor patient prognosis. Mechanistically, IKZF2 overexpression promotes CTCL progression via inhibiting malignant cell apoptosis and may contribute to tumor immune escape by downregulating MHC-II molecules and up-regulating the production of anti-inflammatory cytokine IL-10 by malignant T cells. These results demonstrate the important role of IKZF2 in high-risk CTCL and pave the way for future targeted therapy.

Malignant and Benign T Cells Constituting Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of non-Hodgkin lymphoma, including various clinical manifestations, such as mycosis fungoides (MF) and Sézary syndrome (SS). CTCL mostly develops from CD4 T cells with the skin-tropic memory phenotype. Malignant T cells in MF lesions show the phenotype of skin resident memory T cells (TRM), which reside in the peripheral tissues for long periods and do not recirculate. On the other hand, malignant T cells in SS represent the phenotype of central memory T cells (TCM), which are characterized by recirculation to and from the blood and lymphoid tissues. The kinetics and the functional characteristics of malignant cells in CTCL are still unclear due, in part, to the fact that both the malignant cells and the T cells exerting anti-tumor activity possess the same characteristics as T cells. Capturing the features of both the malignant and the benign T cells is necessary for understanding the pathogenesis of CTCL and would lead to new therapeutic strategies specifically targeting the skin malignant T cells or benign T cells.

Single-Cell and Spatial Analyses Characterize Distinct Subsets of Malignant T Cells in Angioimmunoblastic T Cell Lymphoma

Introduction Angioimmunoblastic T-cell lymphoma (AITL) is the most frequent of nodal peripheral T-cell lymphomas. AITL results from the transformation of T follicular helper (T FH) cells and is characterized by chemo-resistance and poor survival (5-year OS around 30%). Recent data from prospective clinical trials suggest that disease outcome may be impacted by factors other than genomic features, such as the tumor microenvironment (TME) and overall intra-tumoral heterogeneity. Our understanding of AITL intra-tumoral genetic, transcriptional and functional heterogeneity is limited because most molecular data generated so far have come from bulk analyses. Single-cell RNA sequencing (scRNA-seq) enables fine characterization of cell types and functional cell states. When focused on T or B cells, 5'-end scRNA-seq also yields the TCR or BCR sequences that allow tracking clonally related cells. Here we studied the intra-tumor heterogeneity of AITL tumors using integrative scRNA-seq. Methods We analyzed lymph node live cell suspensions from AITL patients (n=10) using droplet-based 10x Genomics 5'-end scRNA-seq. Malignant T cells from 4 AITL samples were also analyzed by FACS index sorting and plate-based 5'-end scRNA-seq to link cell surface phenotype and gene expression profile. We identified malignant T cell clones by intersecting the gene expression and TCR sequencing data, and performed separate focused analyses of TME subsets and malignant T cells. We compared subsets of malignant T cells from all patients using marker gene-based metaclustering to identify AITL T cell states conserved across patients. We explored the genetic heterogeneity of malignant T cells by mapping RHOA G17V mutations and inferring copy number variation (CNV) subclones from scRNA-seq data. In select cases, we performed in situ analysis by immunohistochemistry (IHC) or spatial transcriptomics to characterize the spatial distribution of malignant T cell subsets identified by scRNA-seq. Results Based on gene expression, malignant T cells grouped in patient-specific clusters, while non-malignant T, B and myeloid TME cells from all patients clustered by cell type or cell state. Among TME cells, we identified 7 subsets of B cells (including activated B cells, plasma cells, and one patient-specific monoclonal B cell proliferation), 6 subsets of myeloid cells (including macrophages, conventional and plasmacytoid dendritic cells), and 8 subsets of non-malignant T cells (including activated cytotoxic T lymphocytes (CTL) with clonal expansions). Patient-specific malignant T cells were heterogeneous and divided into several gene-expression based clusters. Metaclustering of malignant T cell subsets identified T central memory (T CM)-like and T FH-like states in 10/10 samples. We also identified in 3/10 samples clusters of CTL-like malignant T cells expressing characteristic marker genes (including NKG7, GNLY, GZMK, PRF1). We observed an intra-sample continuum of gene expression states from quiescent T CM-like to proliferating T FH-like states. T FH-like cells were larger in size and expressed higher levels of surface PD1 and ICOS than T CM-like and CTL-like subsets. We detected the RHOA G17V mutation in malignant T cells of 4/4 mutated cases, with no evidence of subclonal heterogeneity for that mutation. We detected clonal and subclonal CNV in most AITL malignant T cells. CTL-like states were enriched in specific CNV subclones, but the T CM-like to T FH-like continuum was observed in all CNV subclones, suggesting that functional plasticity and subclonal genetic evolution may occur independently. In situ staining of markers for T FH-like (PD1, ICOS, CD200) and CTL-like (GZMK, GZMA) cells showed that T FH-like and CTL-like cells occupied distinct tissue niches within the tumor. In spatial transcriptomics analysis, T FH-like cells mapped to follicular dendritic cell (FDC)-rich areas, while T CM-like cells were associated with T-zone reticular cells. Conclusions Our analyses recapitulate known characteristics of AITL TME, and uncover previously unrecognized heterogeneity among malignant T cells across multiple patients. The distinct gene expression programs, phenotypes, genetics, and locations of T FH-like, T CM-like and CTL-like states suggest that AITL malignant T cells undergo significant functional plasticity and genetic divergence, which could influence response to therapy and overall clinical course. Figure 1 Figure 1. Disclosures Lemonnier: Institut Roche: Research Funding; Gilead: Other: travel grant. Gaulard: Gilead: Consultancy; Innate Pharma: Research Funding; Sanofi: Research Funding; Alderaan: Research Funding; Takeda: Consultancy, Honoraria. Milpied: Institut Roche: Research Funding; Innate Pharma: Research Funding; Bristol Myers Squibb: Research Funding.

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

Background: IL-21 expanded NK cells have high expression of CD16 and have demonstrated antibody-dependent cell-mediated cytotoxicity (ADCC) activity in combination with monoclonal antitumor antibodies (mAb). Mogamulizumab (moga) is a mAb targeting CCR4 that is defucosylated to enhance its binding to CD16, thereby enhancing ADCC of NK cells against targets expressing CCR4. We designed a pilot phase I clinical trial studying this combination in patients with relapsed/refractory (r/r) CTCL and ATLL. The study is soon opening to accrual at the OSU James Cancer Center (NCT04848064). Study is conduced under IND 26888. Preclinical data: Allogeneic NK cells obtained from buffy coat (Red Cross Blood Bank), were expanded for 14 days on CSTX002 feeder cells, cryopreserved, and then thawed and recovered for 48 hours prior to testing. Malignant T-cells were incubated with moga (at 10ng/µl) for 30 minutes prior to co-culture with NK cells and cytotoxicity was determined by the calcein release assay (Somanchi et al, J Vis Exp 2011). Malignant T-cells were obtained from peripheral blood from 3 patients with multiply relapsed CTCL and all have circulating Sezary cells and from CCRF-CEM cell line (T-ALL cell line that expresses CCR4). No significant cytotoxicity was observed with moga alone and significant synergy in cytotoxicity was observed between and moga and NK cells in all 3 patient samples and also CCRF-CEM cell line (figure 1: A and B). Two-fold increase in ADCC was observed with addition of moga to NK cells (p=0.0272; figure 1C) Design: Patients will receive lymphodepleting chemotherapy (Fludarabine/Cyclophosphamide) on days -5 to -3 prior to cell infusion, moga weekly for 4 doses starting on day -7 (prior to the first NK cell infusion) and then every 2 weeks until toxicity or progression. Patients will receive third-party ideal-donor mbIL-21 expanded NK cells once every 2 weeks for 6 total doses (Figure 1D). Donors meeting ideal-donor characteristics from National Marrow Donor Program were identified in collaboration with Be The Match Biotherapies. PBMNC were collected by apheresis, CD3-depleted, expanded for 14 days as previously described, and cryopreserved in ready-to-infuse aliquots. NK cells will be thawed and infused in 2 dosing cohorts; 3x10 7 and 1x10 8 cells/kg in a standard dose-escalation design. Primary endpoint is the maximum tolerated dose of NK cells given in combination with standard-dose moga. Dose-limiting toxicity (DLT) is defined as any steroid refractory graft vs host disease (GVHD), severe NK cell-related toxicities, or other unusual events occurring from D-7 until D+84 post last dose of NK cells. Dose-escalation will proceed in the standard 3+3 fashion. Secondary endpoints include overall response rate (ORR) per ISCL/USCLC/EORTC consensus panel, for CTCL, and international consensus panel, for ATLL and progression-free and overall survival. Correlative endpoints include quality of life impact as captured by Skindex-16 score, serum cytokine levels in blood, persistence of NK cells by chimerism studies, correlation between CCR4 staining of tumor cells in skin and trafficking of NK cells to skin by immunohistochemistry or immunofluorescence in serial skin biopsies and ORR (Figure 1D). Abbreviated eligibility: Eligible patients will be 18 years, or older, with biopsy-proven, measurable, stage IB-IVB relapsed or refractory CTCL or ATLL, progressing on at least one standard chemotherapy. Other eligibility criteria include: ECOG performance status of ≤ 1, no systemic anti-neoplastic therapy within a week or 3 half-lives, adequate laboratory parameters including: absolute neutrophil count ≥1000/mm³, platelet count ≥50,000/mm³, total bilirubin ≤ 2 x upper limit of normal (ULN), AST/ALT ≤ 3 x ULN or ≤ 5 x ULN in patients with documented hepatic involvement by lymphoma, and calculated creatinine clearance ≥ 50 ml/min., disease free of prior malignancies for ≥ 2 years with exception of treated basal cell, squamous cell carcinoma of the skin, or carcinoma in situ of the cervix or breast and life expectancy ≥ 90 days. Patients who were pre-treated with moga, pregnant, HIV positive, with active hepatitis B and C, active CNS involvement, active grade II-IV acute or extensive chronic GVHD or other serious medical comorbid conditions are excluded. Figure 1 Figure 1. Disclosures William: Kyowa Kirin: Consultancy; Incyte: Research Funding; Merck: Research Funding; Dova Pharmaceuticals: Research Funding; Guidepoint Global: Consultancy. de Lima: Miltenyi Biotec: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Devine: Be the Match: Current Employment; Johnsonand Johnson: Consultancy, Research Funding; Orca Bio: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Magenta Therapeutics: Current Employment, Research Funding; Tmunity: Current Employment, Research Funding; Vor Bio: Research Funding; Kiadis: Consultancy, Research Funding. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Lee: Courier Therapeutics: Current holder of individual stocks in a privately-held company; Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. OffLabel Disclosure: Will discuss combination of mogamulizumab and NK cells in a context of a clinical trial

Multimodal single-cell analysis of cutaneous T cell lymphoma reveals distinct sub-clonal tissue-dependent signatures.

Cutaneous T cell lymphoma (CTCL) is a heterogeneous group of mature T cell neoplasms characterized by the accumulation of clonal malignant CD4+ T cells in the skin. The most common variant of CTCL, Mycosis Fungoides, is confined to the skin in early stages but can be accompanied by extracutaneous dissemination of malignant T cells to the blood and lymph nodes in advanced stages of disease. Sézary Syndrome, a leukemic form of disease is characterized by significant blood involvement. Little is known about the transcriptional and genomic relationship between skin and blood residing malignant T cells in CTCL. To identify and interrogate malignant clones in matched skin and blood from leukemic MF and SS patients, we combine T cell receptor clonotyping, with quantification of gene expression and cell surface markers at the single cell level. Our data reveals clonal evolution at a transcriptional and genetic level within the malignant populations of individual patients. We highlight highly consistent transcriptional signatures delineating skin-derived and blood-derived malignant T cells. Analysis of these two populations suggests that environmental cues, along with genetic aberrations, contribute to transcriptional profiles of malignant T cells. Our findings indicate that the skin microenvironment in CTCL promotes a transcriptional response supporting rapid malignant expansion, as opposed to the quiescent state observed in the blood, potentially influencing efficacy of therapies. These results provide insight into tissue-specific characteristics of cancerous cells and underscore the need to address the patients' individual malignant profiles at the time of therapy to eliminate all sub-clones.