Unleashed monocytic engagement in Sézary syndrome during the combination of anti-CCR4 antibody with type I interferon.

Sézary syndrome is an aggressive leukemic expansion of skin derived malignant CD4+ T cells. Drug monotherapy often results in disease relapse due to the heterogenous nature of malignant CD4+ T cells, but how therapies can be optimally combined remains unclear due to limitations in understanding the disease pathogenesis. We identified immunologic transitions that interlink mycosis fungoides (MF) with Sézary syndrome (SS) using single-cell transcriptome analysis in parallel with high-throughput TCR sequencing. Nascent peripheral CD4+ T cells acquired a distinct profile of transcription factors and trafficking receptors that gave rise to antigenically mature Sézary cells. The emergence of malignant CD4+ T cells coincided with the accumulation of dysfunctional monocytes with impaired Fc-dependent phagocytosis, decreased responsiveness to cytokine stimulation and limited repertoire of intercellular interactions with Sézary cells. Type I interferon supplementation when combined with a monoclonal antibody targeting the chemokine receptor type 4 (CCR4), unleashed monocyte induced phagocytosis and eradication of Sézary cells in vitro. In turn, co-administration of interferon- with the FDA-approved anti-CCR4 antibody, mogamulizumab, in patients with SS induced marked depletion of peripheral malignant CD4+ T cells. Importantly, residual CD4+ T cells following Sézary cell ablation lacked any immunologic shifts. These findings collectively unveil an auxiliary role for augmenting monocytic activity during mogamulizumab therapy in the treatment of SS and underscore the importance of targeted combination therapy in this disease.

Deciphering Tumor Cell Evolution in Cutaneous T-Cell Lymphomas: Distinct Differentiation Trajectories in Mycosis Fungoides and Sézary Syndrome.

Cutaneous T-cell lymphomas are a heterogeneous group of neoplasms originating in the skin, with mycosis fungoides (MF) and Sézary syndrome (SS) representing the most common variants. The cellular origin of cutaneous lymphomas has remained controversial owing to their immense phenotypic heterogeneity that obfuscates lineage reconstruction on the basis of classical surface biomarkers. To overcome this heterogeneity and reconstruct the differentiation trajectory of malignant cells in MF and SS, TCR sequencing was performed in parallel with targeted transcriptomics at the single-cell resolution among cutaneous samples in MF and SS. Unsupervised lineage reconstruction showed that Sézary cells exist as a population of CD4+ T cells distinct from those in patch, plaque, and tumor MF. Further investigation of malignant cell heterogeneity in SS showed that Sézary cells phenotypically comprised at least 3 subsets on the basis of differential proliferation potentials and expression of exhaustion markers. A T helper 1-polarized cell type, intermediate cell type, and exhausted T helper 2-polarized cell type were identified, with T helper 1- and T helper 2-polarized cells displaying divergent proliferation potentials. Collectively, these findings provide evidence to clarify the relationship between MF and SS and reveal cell subsets in SS that suggest a possible mechanism for therapeutic resistance.

What Does the Future Hold for Biomarkers of Response to Extracorporeal Photopheresis for Mycosis Fungoides and Sézary Syndrome?

Extracorporeal photopheresis (ECP) is an FDA-approved immunotherapy for cutaneous T-cell lymphoma, which can provide a complete response in some patients. However, it is still being determined who will respond well, and predictive biomarkers are urgently needed to target patients for timely treatment and to monitor their response over time. The aim of this review is to analyze the current state of the diagnostic, prognostic, and disease state-monitoring biomarkers of ECP, and outline the future direction of the ECP biomarker discovery. Specifically, we focus on biomarkers of response to ECP in mycosis fungoides and Sézary syndrome. The review summarizes the current knowledge of ECP biomarkers, including their limitations and potential applications, and identifies key challenges in ECP biomarker discovery. In addition, we discuss emerging technologies that could revolutionize ECP biomarker discovery and accelerate the translation of biomarker research into clinical practice. This review will interest researchers and clinicians seeking to optimize ECP therapy for cutaneous T-cell lymphoma.

CD5+ Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type, Presenting as an Asymptomatic Nodule.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT), is a rare and aggressive variant of primary cutaneous lymphoma that typically expresses B cells as well as MUM1/IRF4, BCL2, and FOXP1, whereas BCL6 may be present or undetectable. We present a case of CD5+ PCDLBCL-LT presenting as a 6 mm pink-bluish nodule on the mid-left thigh, which was concerning for basal cell carcinoma. The histological examination reveals the presence of an intradermal proliferation of large, atypical CD5+, CD20+ BCL2+, BCL6+, MUM-1+, and Cyclin-D1+ lymphocytes in a nodular, diffuse interstitial and perivascular distribution. Because the patient presented with a small, single nodule, the systemic treatment of multiagent chemotherapy was avoided and localized electron beam radiation therapy with rituximab was initiated instead, achieving complete response. Early identification of PCDLBCL-LT is key for maximal therapeutic benefit and prognosis; it is important to consider PCDLBCL-LT on the differential when evaluating small, single nodules on the lower extremities of elderly patients.

Blockade of the Immune Checkpoint CD47 by TTI-621 Potentiates the Response to Anti-PD-L1 in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an incurable and cosmetically disfiguring disease associated with microenvironmental signals. We investigated the effects of CD47 and PD-L1 immune checkpoint blockades, as a strategy for targeting both innate and adaptive immunity. CIBERSORT analysis identified the immune-cell composition in the CTCL tumor microenvironment and the immune checkpoint expression profile for each immune-cell gene cluster from CTCL lesions. We investigated the relationship between MYC and CD47 and PD-L1 expression and found that MYC short hairpin RNA knockdown and MYC functional suppression by TTI-621 (SIRPαFc) and anti-PD-L1 (durvalumab) in CTCL cell lines reduced the expression of CD47 and PDL1 mRNA and protein as measured by qPCR and flow cytometry, respectively. In vitro, blockade of the CD47-SIRPα interaction with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing in a mixed leucocyte reaction. Moreover, TTI-621 synergized with anti-PD-L1 in macrophages reprogram to M1-like phenotypes and inhibited CTCL cell growth. These effects were mediated by cell death-related pathways, including apoptosis, autophagy, and necroptosis. Collectively, our findings show that CD47 and PD-L1 are critical regulators of immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy for CTCL.

Targeting the CD47-SIRPα Axis: Present Therapies and the Future for Cutaneous T-cell Lymphoma.

The loss of CD47 on aging cells serves as a signal to macrophages to eliminate the target. Therefore, CD47 is a "do-not-eat-me" sign preventing macrophagal phagocytosis via interaction with its ligand SIRPα. Malignant lymphocytes of mycosis fungoides and Sézary syndrome express CD47 highly, thus, being ideal candidates for targeted anti-CD47 therapies. The classes of current anti-CD47-SIRPα therapeutic molecules present in a large variety and include monoclonal antibodies against CD47 and SIRPα, bioengineered SIRPα proteins, miRNAs, and bispecific antibodies. We provided a detailed analysis of all available investigational drugs in a contest of cutaneous T-cell lymphoma. A combination of blockade of the CD47-SIRPα axis and secondary targets in the tumor microenvironment (TME) may improve the clinical efficacy of current immunotherapeutic approaches. We evaluated the possible combination and outlined the most promising one.

Repetitive expanded T-cell receptor clonotypes impart the classic T helper 2 Sézary cell phenotype.

Six out of 12 Sézary patients shared one clonotype (TRAV13-1*01-TRAJ49*01-TRBV20-1*01-TRBJ2-3*01). TRBV20-1*01 (also known as Vb2) that binds toxic shock syndrome toxin-1 was utilized by Sézary cells among half of the cohort, which would be expected for a common unifying origin.

Exhausted Markers in Cutaneous T-Cell Lymphoma: The Face that Launched a Thousand Ships.

Immune-modulatory therapies are widely appreciated to rejuvenate host antitumor immunity and improve mortality in solid-organ cancers. Targeting the exhausted markers such as PD-1, CTLA4, TIM3, LAG3 are particularly attractive owing to the activation of the immune response. However, their role in cutaneous T-cell lymphomas is less defined owing to the expression of those exhausted markers on both nonmalignant and malignant lymphocytes. In a new article of the Journal of Investigative Dermatology, Han et al. (2021) showed that microRNAs in malignant T cells could regulate the expression of PD-1, CTLA4, TIM3, and LAG3 and simultaneously mediate evasion from immune surveillance. These findings get us one step closer in our further investigation of whether those molecules could be targeted therapeutically.

Understanding Cell Lines, Patient-Derived Xenograft and Genetically Engineered Mouse Models Used to Study Cutaneous T-Cell Lymphoma.

Cutaneous T cell lymphoma (CTCL) is a spectrum of lymphoproliferative disorders caused by the infiltration of malignant T cells into the skin. The most common variants of CTCL include mycosis fungoides (MF), Sézary syndrome (SS) and CD30+ Lymphoproliferative disorders (CD30+ LPDs). CD30+ LPDs include primary cutaneous anaplastic large cell lymphoma (pcALCL), lymphomatoid papulosis (LyP) and borderline CD30+ LPD. The frequency of MF, SS and CD30+ LPDs is ~40-50%, <5% and ~10-25%, respectively. Despite recent advances, CTCL remains challenging to diagnose. The mechanism of CTCL carcinogenesis still remains to be fully elucidated. Hence, experiments in patient-derived cell lines and xenografts/genetically engineered mouse models (GEMMs) are critical to advance our understanding of disease pathogenesis. To enable this, understanding the intricacies and limitations of each individual model system is highly important. Presently, 11 immortalized patient-derived cell lines and different xenograft/GEMMs are being used to study the pathogenesis of CTCL and evaluate the therapeutic efficacy of various treatment modalities prior to clinical trials. Gene expression studies, and the karyotyping analyses of cell lines demonstrated that the molecular profile of SeAx, Sez4, SZ4, H9 and Hut78 is consistent with SS origin; MyLa and HH resemble the molecular profile of advanced MF, while Mac2A and PB2B represent CD30+ LPDs. Molecular analysis of the other two frequently used Human T-Cell Lymphotropic Virus-1 (HTLV-1)+ cell lines, MJ and Hut102, were found to have characteristics of Adult T-cell Leukemia/Lymphoma (ATLL). Studies in mouse models demonstrated that xenograft tumors could be grown using MyLa, HH, H9, Hut78, PB2B and SZ4 cells in NSG (NOD Scid gamma mouse) mice, while several additional experimental GEMMs were established to study the pathogenesis, effect of drugs and inflammatory cytokines in CTCL. The current review summarizes cell lines and xenograft/GEMMs used to study and understand the etiology and heterogeneity of CTCL.

The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides.

CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.

Cutaneous metastasis of primary diffuse large B-cell lymphoma of the central nervous system developing 4 years after complete remission: Diagnosis confirmed by comparison of clones.

B-cell lymphoma of the central nervous system (CNS) is a rare malignancy with diffuse large B-cell lymphoma (DLBCL) variant being most common. Although DLBCL has a high propensity to relapse locally within the CNS, only a few cases of cutaneous metastasis have been described in the literature. We present a unique case of cutaneous metastasis of a primary DLBCL of the CNS in a 79-year-old man who was in clinical remission for 4 years until presenting with a lesion in the left adrenal gland and cutaneous nodules on the left flank. Skin biopsy specimen revealed a diffuse dermal infiltrate of atypical B-cell lymphocytes with expression of CD20, BCL-2, BCL-6, and MUM-1, suggestive of DLBCL. For differentiation between another primary or a recurrent process, immunoglobulin kappa (IgK) light chain gene rearrangement was performed and demonstrated that the DLBCL of the skin and CNS were of the same clonal origin. Restaging computerized tomography after initiating chemotherapy and daily ibrutinib showed complete resolution of the left adrenal mass and resolving cutaneous lesions. Our case demonstrates the rare, late cutaneous metastasis of DLBCL of the CNS and highlights the importance of genetic testing for the distinction between the primary and secondary lesions.

Clinical Response to Anti-CD47 Immunotherapy Is Associated with Rapid Reduction of Exhausted Bystander CD4+ BTLA+ T Cells in Tumor Microenvironment of Mycosis Fungoides.

Cancer progression in mycosis fungoides, the most common form of cutaneous T-cell lymphoma, occurs in a predictable, sequential pattern that starts from patches and that evolves to plaques and later to tumors. Therefore, unlocking the relationship between the microarchitecture of mycosis fungoides and the clinical counterparts of that microstructure represents important steps for the design of targeted therapies. Using multispectral fluorescent imaging, we show that the progression of mycosis fungoides from plaque to tumor parallels the cutaneous expansion of the malignant CD4+ T cells that express TOX. The density of exhausted BTLA+ CD4+ T cells around malignant CD4+TOX+ cells was higher in tumors than it was in plaques, suggesting that undesired safeguards are in place within the tumor microenvironment that prevent immune activation and subsequent cancer eradication. Overriding the CD47 checkpoint with an intralesional SIRPαFc fusion decoy receptor induced the resolution of mycosis fungoides in patients that paralleled an amplified expansion of NK and CD8+ T cells in addition to a reduction of the exhausted BTLA+ CD4+ T cells that were engaged in promiscuous intercellular interactions. These therapeutic benefits of the CD47 blockade were further unleashed by adjuvant interferon-α, which stimulates cytotoxic cells, underscoring the importance of an inflamed microenvironment in facilitating the response to immunotherapy. Collectively, these findings support CD47 as a therapeutic target in treating mycosis fungoides and demonstrate a synergistic role of interferon-α in exploiting these clinical benefits.

Intralesional TTI-621, a novel biologic targeting the innate immune checkpoint CD47, in patients with relapsed or refractory mycosis fungoides or Sézary syndrome: a multicentre, phase 1 study.

BACKGROUND: Intravenous TTI-621 (SIRPα-IgG1 Fc) was previously shown to have activity in relapsed or refractory haematological malignancies. This phase 1 study evaluated the safety and activity of TTI-621 in patients with percutaneously accessible relapsed or refractory mycosis fungoides, Sézary syndrome, or solid tumours. Here we report the clinical and translational results among patients with mycosis fungoides or Sézary syndrome. METHODS: This multicentre, open-label, phase 1 study was conducted at five academic health-care and research centres in the USA. Eligible patients were aged 18 years or older; had injectable, histologically or cytologically confirmed relapsed or refractory cutaneous T-cell lymphoma (CTCL) or solid tumours; Eastern Cooperative Oncology Group performance status of 2 or less; and adequate haematological, renal, hepatic, and cardiac function. TTI-621 was injected intralesionally in a sequential dose escalation (cohorts 1-5; single 1 mg, 3 mg, or 10 mg injection or three 10 mg injections weekly for 1 or 2 weeks) and in expansion cohorts (cohorts 6-9; 2 week induction at the maximum tolerated dose; weekly continuation was allowed). In cohort 6, patients were injected with TTI-621 in a single lesion and in cohort 7, they were injected in multiple lesions. In cohort 8, TTI-621 was combined with pembrolizumab 200 mg injections per product labels. In cohort 9, TTI-621 was combined with the standard labelled dose of subcutaneous pegylated interferon alpha-2a 90 µg. The primary endpoint was the incidence and severity of adverse events. The study is registered with ClinicalTrials.gov, NCT02890368, and was closed by the sponsor to focus on intravenous studies with TTI-621. FINDINGS: Between Jan 30, 2017, and March 31, 2020, 66 patients with mycosis fungoides, Sézary syndrome, other CTCL, or solid tumours were screened, 35 of whom with mycosis fungoides or Sézary syndrome were enrolled and received intralesional TTI-621 (escalation, n=13; expansion, n=22). No dose-limiting toxicities occurred; the maximum tolerated dose was not established. In the dose expansion cohorts, the maximally assessed regimen (10 mg thrice weekly for 2 weeks) was used. 25 (71%) patients had treatment-related adverse events; the most common (occurring in ≥10% of patients) were chills (in ten [29%] patients), injection site pain (nine [26%]), and fatigue (eight [23%]). No treatment-related adverse events were grade 3 or more or serious. There were no treatment-related deaths. Rapid responses (median 45 days, IQR 17-66) occurred independently of disease stage or injection frequency. 26 (90%) of 29 evaluable patients had decreased Composite Assessment of Index Lesion Severity (CAILS) scores; ten (34%) had a decrease in CAILS score of 50% or more (CAILS response). CAILS score reductions occurred in adjacent non-injected lesions in eight (80%) of ten patients with paired assessments and in distal non-injected lesions in one additional patient. INTERPRETATION: Intralesional TTI-621 was well tolerated and had activity in adjacent or distal non-injected lesions in patients with relapsed or refractory mycosis fungoides or Sézary syndrome, suggesting it has systemic and locoregional abscopal effects and potential as an immunotherapy for these conditions. FUNDING: Trillium Therapeutics.

Research Techniques Made Simple: Skin-Targeted Drug and Vaccine Delivery Using Dissolvable Microneedle Arrays.

Skin-targeted drug delivery is broadly employed for both local and systemic therapeutics and is an important tool for discovery efforts in cutaneous biology. Recently, emerging technologies support efforts toward skin-targeted biocargo delivery for local and systemic therapeutic benefit. Effective targeting of bioactive molecules, including large (molecular weight > 500 Da) or complex (hydrophilic and charged) molecules, to defined cutaneous microenvironments is intrinsically challenging owing to the protective barrier function of the skin. Dissolvable microneedle arrays (MNAs) have proven to be a promising technology to address the unmet need for controlled, minimally invasive, and reliable delivery of a wide range of biocargos to the skin. In this paper, we describe the unique properties of the skin that make it an attractive target for vaccine delivery, for immune-modulating therapies, and for systemic drug delivery and the structural characteristics of the skin that present obstacles to efficient intracutaneous and transdermal delivery of bioactive molecules. We provide an overview of MNA fabrication and the characteristics and mechanisms of dissolvable MNA cargo delivery to the cutaneous microenvironment. We present a representative example of a clinical application of MNAs and discuss future directions for MNA development and applications.

Randomized phase 3 ALCANZA study of brentuximab vedotin vs physician's choice in cutaneous T-cell lymphoma: final data.

The primary analysis of the phase 3 ALCANZA trial showed significantly improved objective responses lasting ≥4 months (ORR4; primary endpoint) and progression-free survival (PFS) with brentuximab vedotin vs physician's choice (methotrexate or bexarotene) in CD30-expressing mycosis fungoides (MF) or primary cutaneous anaplastic large-cell lymphoma (C-ALCL). Cutaneous T-cell lymphomas often cause pruritus and pain; brentuximab vedotin improved skin symptom burden with no negative effects on quality of life. We report final data from ALCANZA (median follow-up, 45.9 months). Adults with previously treated CD30-expressing MF/C-ALCL were randomly assigned to brentuximab vedotin (n = 64) or physician's choice (n = 64). Final data demonstrated improved responses per independent review facility with brentuximab vedotin vs physician's choice: ORR4; 54.7% vs 12.5% (P < .001); complete response, 17.2% vs 1.6% (P = .002). Median PFS with brentuximab vedotin vs physician's choice was 16.7 months vs 3.5 months (P < .001). Median time to the next treatment was significantly longer with brentuximab vedotin than with physician's choice (14.2 vs 5.6 months; hazard ratio, 0.27; 95% confidence interval, 0.17-0.42; P < .001). Of 44 patients in the brentuximab vedotin arm who experienced any-grade peripheral neuropathy, (grade 3, n = 6; grade 4, n = 0), 86% (38 of 44) had complete resolution (26 of 44) or improvement to grades 1 and 2 (12 of 44). Peripheral neuropathy was ongoing in 18 patients (all grades 1-2). These final analyses confirm improved, clinically meaningful, durable responses and longer PFS with brentuximab vedotin vs physician's choice in CD30-expressing MF or C-ALCL. This trial was registered at https://www.clinicaltrials.gov as #NCT01578499.