Development Of Cutaneous T-cell Lymphoma Research Articles

Cutaneous T-Cell Lymphoma and Microbiota: Etiopathogenesis and Potential New Therapeutic Targets.

To review the scientific literature related to human microbiota and cutaneous T-cell lymphoma. Methodology. An exploratory and systematic review of the articles retrieved from the bibliographic databases MEDLINE (PubMed), Embase, The Cochrane Library, and Scopus, published in the last 10 years with the following descriptors: "lymphoma, T-cell, cutaneous," "microbiota," "Mycosis Fungoides," "Sézary Syndrome," "lymphoma, primary cutaneous anaplastic large cell," "Lymphomatoid Papulosis" and "Microbiota," "microbiota," "Microbial Community," and "Microbial Communities." Of the 87 references retrieved, after applying the inclusion and exclusion criteria, 21 articles were selected. Most studies linking cutaneous T-cell lymphoma and the microbiota focus on the cutaneous microbiome, with Staphylococcus aureus being the main related agent. Skin colonization by this bacterium could be involved in the hyperactivation of the STAT3 inflammatory pathway and in the overproduction of IL-17, both of which are widely related to the development of more aggressive and advanced forms of cutaneous T-cell lymphoma. We also found evidence of a possible relationship between intestinal dysbiosis and the development of cutaneous T-cell lymphoma, observing a decrease in taxonomic variability and an increase in certain genera such as Prevotella in the intestinal microbiome of patients with cutaneous T-cell lymphoma. The possible etiopathogenic mechanism underlying this relationship could be explained by an increase in systemic cytokine release, promoting the hyperactivation of STAT3 at the skin level. There appears to be a relationship between cutaneous T-cell lymphoma and the cutaneous and intestinal microbiome, as well as a possible pathophysiological pathway involved. The possible modulation of the cutaneous and intestinal microbiome or the action on the signaling inflammatory pathway, using pharmacological tools such as JAK inhibitors or IL-17 inhibitors in the latter case, could open the possibility for future therapeutic studies for cutaneous T-cell lymphoma.

The Complex Role of Infectious Agents in Human Cutaneous T-Cell Lymphoma Pathogenesis: From Candidate Etiological Factors to Potential Therapeutics.

Cutaneous T-cell lymphoma (CTCL) is a devastating, potentially fatal T-lymphocyte malignancy affecting the skin. Despite all efforts, the etiology of this disease remains unknown. Infectious agents have long been suspected as factors or co-factors in CTCL pathogenesis. This review deals with the panel of bacterial and viral pathogens that have been investigated so far in an attempt to establish a potential link between infection/carriage and CTCL development. A special focus is given to a recently discovered human protoparvovirus, namely the cutavirus (CutaV), which has emerged as a plausible CTCL etiological agent. Available evidence in support of this hypothesis as well as alternative interpretations and uncertainties raised by some conflicting data are discussed. The complexity and multifacetedness of the Parvoviridae family of viruses are illustrated by presenting another protoparvovirus, the rat H-1 parvovirus (H-1PV). H-1PV belongs to the same genus as the CutaV but carries considerable potential for therapeutic applications in cutaneous lymphoma.

Risk of Cutaneous TCell Lymphoma with Psoriasis Biologic Therapies.

The risk of developing cutaneous Tcell lymphoma (CTCL) in patients using psoriasis biologics has not been well characterized. The goals of this review were to investigate the incidence of CTCL in patients with psoriasis receiving biologic therapy in clinical trials and psoriasis registries, and to review cases of CTCL and biologic use reported in scientific publications. The US National Library of Medicine clinical trials database (clinicaltrials.gov) was queried to identify phase3 and 4 clinical trials of the 12 biologic agents currently FDA approved for psoriatic disease. The incidence of CTCL in these trials was examined and summarized. To examine the incidence of CTCL in psoriasis registries, a Medline search was conducted. Finally, we performed a systematic review of CTCL cases reported in the literature. Only two cases of CTCL were reported in 35,801 subjects with psoriasis receiving a biologic agent in the active arm of 108 psoriasis phase3 clinical trials. One of these CTCL cases was determined by the investigator to be CTCL misdiagnosed as psoriasis prior to randomization. No cases of CTCL were reported in 5440subjects with psoriasis in 34 phase4 clinical trials. Only one case of CTCL was identified in 34,111 registry subjects. In the literature, tumor necrosis factor (TNF) inhibitors had the highest number of reported cases of CTCL (34 cases), followed by interleukin (IL)-17 inhibitors (7 cases), and IL-12/23 inhibitors (6 cases). No cases of CTCL were found to be reported with IL-23 inhibitors. Our findings indicate that the development of CTCL is rare in the setting of psoriasis biologic use. Of the limited number of cases of CTCL found, most were in the setting of TNF inhibitor use and no cases of CTCL were reported in the setting of IL-23 inhibitor use.

Dupilumab-Associated Lymphoid Reactions in Patients With Atopic Dermatitis

Since the increased use of dupilumab for atopic dermatitis (AD) in daily practice, several cases have been reported on the development of cutaneous T-cell lymphomas (CTCL) and lymphoid infiltrates. To provide insight in the clinical and histopathologic features of patients with AD clinically suspected for CTCL during dupilumab treatment. This retrospective observational case series included adult (≥18 years) patients with AD treated with dupilumab between October 2017 and July 2022 at the University Medical Center Utrecht in the Netherlands. Relevant patient, disease, and treatment characteristics were evaluated. Skin biopsies before, during, and after treatment were collected and reassessed. Fourteen patients (54.5% male) with a median (IQR) age of 56 (36-66) years suspected for CTCL with deterioration of symptoms during dupilumab treatment were included. Of 14 patients, 3 were retrospectively diagnosed with preexistent mycosis fungoides (MF). Eleven patients with AD were eventually diagnosed with a lymphoid reaction (LR). These patients showed MF-like symptoms; however, histopathologic findings were different, and included sprinkled distribution of small hyperchromatic lymphocytes in the upper epidermal section, a dysregulated CD4:CD8 ratio, and CD30 overexpression, without loss of CD2/CD3/CD5. The median time to clinical worsening was 4.0 months (IQR, 1.4-10.0). Posttreatment biopsies showed complete clearance of the LR in all patients. This study found that dupilumab treatment can cause a reversible and benign LR, which mimics a CTCL, though has distinctive histopathologic features.

A CASE REPORT: TRANSFORMATION OF ERYTHRODERMIC PSORIASIS TO CUTANEOUS T-CELL LYMPHOMA

Background: Erythrodermic psoriasis (EP) is a severe type of psoriasis, a chronic inflammatory disease characterized by general erythematous condition with thick scale all around the body. The treatment of EP consists systemic medications like cyclosporine or methotrexate and biologic agents, which lately suggested related to the development of cutaneous T-cell lymphoma (CTCL) in severe psoriasis patients.
 Case Report: A 45-year old, Javanese male was diagnosed previously with EP that transformed to CTCL. His previous history, he was histologically confirmed as psoriasis vulgaris since 2014 and was treated regularly with methotrexate. After 5 years, he gradually suffered several episodes of erythrodermic condition and was diagnosed with erythrodermic psoriasis with unusual pruritic hyperkeratotic plaques and ulcers on his trunks without any general lymph nodes enlargement. Peripheral blood smear showed Sezary cell and histopathology result confirmed CTCL. He was treated with combination of radiotherapy, phototherapy and topical regiments with a good result and acceptable.
 Discussion: Several studies suggested that severe psoriasis had increased the susceptibility of CTCL, especially with the use of cyclosporine, methotrexate or biologic agents as the treatment. In this case, methotrexate might play role in the development of CTCL or enhanced the transformation of EP to CTCL. Combination of localized radiation therapy, NBUVB and topical therapy gave a quiet good result for the skin condition and increased patient’s quality of life.
 Conclusion: Erythrodermic psoriasis might transform or develop to CTCL which in this case was probably related to the use of methotrexate.
 

Integrated transcriptome and trajectory analysis of cutaneous T-cell lymphoma identifies putative precancer populations.

The incidence of cutaneous T-cell lymphoma (CTCL) increases with age, and blood involvement portends a worse prognosis. To advance our understanding of the development of CTCL and identify potential therapeutic targets, we performed integrative analyses of paired single-cell RNA and T-cell receptor (TCR) sequencing of peripheral blood CD4+ T cells from patients with CTCL to reveal disease-unifying features. The malignant CD4+ T cells of CTCL showed highly diverse transcriptomic profiles across patients, with most displaying a mature Th2 differentiation and T-cell exhaustion phenotype. TCR-CDR3 peptide prediction analysis suggested limited diversity between CTCL samples, consistent with a role for a common antigenic stimulus. Potential of heat diffusion for affinity-based trajectory embedding transition analysis identified putative precancerous circulating populations characterized by an intermediate stage of gene expression and mutation level between the normal CD4+ T cells and malignant CTCL cells. We further revealed the therapeutic potential of targeting CD82 and JAK that endow the malignant CTCL cells with survival and proliferation advantages.

851 Cancer associated fibroblasts in different T-stage lesions of cutaneous T-cell lymphoma

The tumor microenvironment (TME) has been shown to play an important role in tumor initiation, development, and metastasis. Among all TME factors, cancer associated fibroblasts (CAF) have been suggested to play a key role. Two studies have reported that fibroblasts from cutaneous T-cell lymphoma (CTCL) lesions, mycosis fungoides (MF) and/or Sézary syndrome (SS), were hyper-activated and exerted a pro-tumorigenic activity on CTCL cells in vitro. But much regarding CAFs in development of CTCL remains to be understood.

LB884 Integrated transcriptome and trajectory analysis of cutaneous T-cell lymphoma identifies putative precancer populations

Cutaneous T-cell lymphoma (CTCL) incidence increases with age, and blood involvement portends a worse prognosis. To advance our understanding of CTCL development and identify potential therapeutic targets, we performed integrative analyses of paired single-cell RNA and TCR sequencing of peripheral blood CD4+ T-cells from CTCL patients to reveal disease unifying features. The malignant CD4+ T-cells of CTCL show highly diverse transcriptomic profiles across patients, with most displaying a mature Th2 differentiation and T-cell exhaustion phenotype.

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 [Display omitted] DisclosuresLopez: 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.

Prec-O2-01 - 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.

Bio-O1-08 - PLCγ1/PKCθ and its downstream effectors in cutaneous T-cell lymphoma development and progression

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas (CTCL). In the last few years, the use of Next Generation Sequencing (NGS) approaches has revealed a rich landscape of genomic alterations potentially influencing the biology of these entities. In this regard, multiple genetic and biological evidences point at a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, to control the biology of CTCLs. In addition, activated STAT3 is associated with clinical progression, although the specific mechanisms responsible for this have not been fully elucidated. Here we studied PLCγ1-dependent mechanisms that can activate JAK/STAT and control tumor growth and progression. Downstream of PLCγ1, we identified PKCθ (a specific T cell PKC isoform) to mediate JAK/STAT3 activation and control CTCL cell proliferation and viability. In vivo, PKCθ blockage in CTCL cells, impaired tumor development and cells spreading to the liver and the lungs. In light of these results, we sought to identify specific PKCθ downstream effectors, that in addition to JAK/STATs, could help understanding its biological role in CTCL. To this end, we studied PKCθ-dependent transcriptome as well as its protein interactome in CTCL cells. This approach helped detecting a number of hits that can potentially explain STAT3 phosphorylation in CTCL cells (i.e. RACK1), and highlight a number of oncogenic activities controlled by PKCq, like mitochondrial activity (i.e. ASK1, HSPA9) and cytoskeletal regulation (i.e. ARHGAP-19 or CCT4, 5). As a result, this dual approach helped identifying a number of protein effectors and target genes that can participate in important CTCL activities like for example cytokine signaling, mitochondrial activity, TP53, and actin cytoskeleton dynamics. Finally, we took advantage of Nanostring to study the expression of a selection of PKCθ target genes, in a cohort of 81 human MF samples and control inflammatory dermatoses. In this context, PRKCQ (PKCθ) gene expression was significantly upregulated in MF cases vs. controls. Moreover, the expression of target genes like PRKACB (encoding for PKA isoform B) or the lymphocyte specific actin binding LCP1 and LCP1, positively correlated with that of PRKCQ expression in MF cases. On the other hand, genes like FGFR3 and RHOB showed a significant yet inverse correlation with PRKCQ in robust agreement with the data generated in CTCL cells. Overall, the results show a PKCθ gene signature that is significant and differentially expressed amongst different MF cases, at different stages of the disease. Moreover, this signature highlights novel CTCL disease mechanisms with translational potential in diagnosis and targeted therapy. In summary, we have uncover a series of PKCθ-dependent effectors/routes that can play a central role in the biology of CTCLs and help to develop novel approaches to diagnose and treat these malignancies.

Bio-P-03 - Bioinformatics insights in etiopathogenesis, diagnosis and therapy of cutaneous T-cell lymphoma

<h3>Introduction:</h3> The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains poorly understood and the histologic diagnosis of early MF is one of the most vexing problems in dermatopathology. Our aim was to apply state-of-the-art bioinformatic tools in order to unravel the mechanisms of CTCL pathobiology, to address new therapeutic possibilities as well as to identify potential biomarkers. <h3>Materials and methods:</h3> Datasets selection: we searched the GEO (http://www.ncbi.nlm.nih.gov/geo/) for gene (GSE143382) and miRNA (GSE109421) expression datasets containing early-stage MF samples and reactive skin lesions (inflammatory dermatosis) as control. Differential expression data analysis: was performed in R programming language using the LIMMA R package. Differentially expressed genes (DEGs) were selected by applying selection thresholds of adjusted p-value < 0.05 and absolute (log2FC) ≥1 (i.e. FC ≥2 or FC ≤0.5). For the case of differentially expressed miRNAs (DEMs) we used as selection criteria the p-value <0.05 and the FC ≥1.2 for the overexpressed miRNAs and FC ≤0.83 for the under-expressed ones. Pathway Enrichment Analysis using DEGs: was performed using the Enrichr, a web-based tool for analyzing gene sets that returns any enrichment of common annotated biological features (https://maayanlab.cloud/Enrichr/). Pathway Enrichment Analysis using DEMs: was performed using the DIANA-mirPath which is a miRNA pathway analysis web-server, which can utilize experimentally validated miRNA interactions derived from DIANA-TarBase v6.0. The enrichment was obtained using information from KEGG Human and the most significantly enriched pathways were selected based on the p-value <0.05. <h3>Results:</h3> Our bioinformatics analysis detected: A list of DEGs in CTCL which are associated with increased cell proliferation decreased apoptosis, increased Th2 differentiation and immune activation leading to generation of malignant CD4+ T-cells clones, propagating the disease. A list of DEMs in CTCL. A more restricted set of overexpressed miRNAs (miR-26a, miR-92a, miR-106b, miR-142, miR-146a, miR-155, miR-181a, miR-222 and miR-494) were selected and could be therefore used as biomarkers. A number of pathways that have been extracted from the genes and miRNAs implicated in the pathogenesis of CTCL and can enlighten its etiopathogenesis. Fourteen percent (14%) of the pathways were common in both analyses including TGF-beta signaling pathway, Pathways in cancer, PI3K-Akt signaling pathway and p53 signaling pathway. <h3>Conclusion:</h3> In this project, the tools of bioinformatics were used as a stepping-stone in a combined approach highlighting differentially expressed genes and miRNAs as well as the implicated pathways involved in CTCL development. These findings formed a resource of genes and miRNAs that will be further tested in vitro and in vivo in order to identify the etiopathogenesis of the disease, discover biomarkers for diagnosis and highlight new therapeutic targets.

Targeting the non-ATP-binding pocket of the MAP kinase p38γ mediates a novel mechanism of cytotoxicity in cutaneous T-cell lymphoma (CTCL).

We describe here for the first time a lipid‐binding‐domain (LBD) in p38γ mitogen‐activated protein kinase (MAPK) involved in the response of T cells to a newly identified inhibitor, CSH71. We describe how CSH71, which binds to both the LBD and the ATP‐binding pocket of p38γ, is selectively cytotoxic to CTCL Hut78 cells but spares normal healthy peripheral blood mononuclear (PBMC) cells, and propose possible molecular mechanisms for its action. p38γ is a key player in CTCL development, and we expect that the ability to regulate its expression by specifically targeting the lipid‐binding domain will have important clinical relevance. Our findings characterize novel mechanisms of gene regulation in T lymphoma cells and validate the use of computational screening techniques to identify inhibitors for therapeutic development.

Abstract 2759: Reprogramming of PD1+ M2-like tumor-associated macrophages with anti-PD-L1 and Lenalidomide in cutaneous T cell lymphoma

Abstract M2-like tumor-associated macrophages (TAMs) are abundant and influence cutaneous T cell lymphoma (CTCL) development by inducing immunosuppression. Although it is well established that depletion of M2-like TAMs delays CTCL development, little is known about the underlying mechanisms that shape programmed cell death 1+ (PD1+) M2-like TAMs phenotype in CTCL. Here we identified PD1+ M2-like TAMs accumulated in CTCL lesional skin. Moreover, RNA-seq analysis of human CTCL tissues revealed an up-regulation of the TLR/NF-κB and JAK/STAT signaling pathways. In vitro, PD1+ TAMs exhibited an M2-like profile, with a significant increase in the expression of CD163, CD206, and IL-10, and a clear decrease in the expression of CD80, IL-1β, CXCL-10, and CXCL-11, and the activation of TLR/NF-κB and JAK/STAT signaling pathways. To determine whether PD1+ M2-like TAMs could be reprogrammed, anti-PD-L1 (durvalumab) and lenalidomide were used for treatment of MyLa-conditioned media induced human peripheral blood monocyte-derived PD1+ M2-like TAMs. The results show that anti-PD-L1 and lenalidomide synergistically reshaped M2-like TAMs to M1-like TAMs in vitro through ablation of the TLR/NF-κB and JAK/STAT signaling pathways, which was linked with functional changes in phagocytic activity and cell migration. In conclusion, TLR/NF-κB and JAK/STAT signaling pathways may drive PD1+ M2-like TAMs programming in the CTCL tumor microenvironment, anti-PD-L1 and lenalidomide may reshape PD1+ M2-like TAMs and induce functional changes through ablation of these specific pathways in CTCL. Corresponding author: Christiane Querfeld, MD, PhD. Div. of Dermatology and Beckman Research Institute, City of Hope. 1500 E. Duarte Road, Duarte, CA 91010. Email: cquerfeld@coh.org Phone: 626-634-4436 Fax: 626-218-6190 Citation Format: Zhen Han1,5, Chingyu Su1,5, Xiwei Wu2,5, Hanjun Qin2,5, Steven T. Rosen3,5, Christiane Querfeld1,3,4,5. Reprogramming of PD1+ M2-like tumor-associated macrophages with anti-PD-L1 and Lenalidomide in cutaneous T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2759.

Therapeutic Targeting of Microrna-214 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature CD4+ T-cells that proliferate in the skin. With advanced disease some patients may experience progression to an aggressive form in which neoplastic cells disseminate to the blood. A hallmark feature in the development and progression of CTCL is the global dysregulation of epigenetic modifications resulting in aberrant gene expression, increased expression of oncogenes, and silencing of tumor suppressors. Therapeutic strategies which target the epigenome have been effective in patients with advanced-stage disease, however short duration of response and development of resistance remain important clinical problems. In this study, we sought to probe the aberrant epigenetic modifications in malignant T-cells and identify potential novel targets for improved therapies.Oncogenic microRNA (miR)-214 has been shown to be significantly upregulated in patients with advanced CTCL, and this overexpression is associated with worse outcomes. The mechanisms by which miR-214 is upregulated in CTCL are unknown. Utilizing CTCL patient-derived cell lines, primary patient samples, and the recently characterized interleukin-15 (IL-15) transgenic mouse model of CTCL, we describe the regulation of miR-214 expression by bromodomain-containing protein 4 (BRD4) as well as aberrantly-expressed TWIST1. We also describe the efficacy of a specific inhibitor of miR-214 in vivo.We recently described increased global binding occupancy by BRD4 in CTCL patients, resulting in oncogene overexpression (Kohnken, R. et al. Blood 128:1097, 2016). Here, we show that BRD4 binding occupancy in CTCL patient cells is increased at the promoter region of miR-214. We confirm significant overexpression of miR-214 in CD4+ T-cells in CTCL patients) compared to healthy donors (Figure 1A, mean ± SEM of expression in CTCL patients vs healthy donor CD4+ cells= 10.18 ± 1.22, n=7 vs 1.03 ± 0.06, n=6, unpaired t-test, p=0.0028). Specific inhibition of BRD4 by small molecule JQ1 significantly decreased miR-214 in both patient CD4+ T-cells (0.58 ± 0.008 compared to 1.00 ± 0.003 in control-treated cells, p<0.0001), and in CTCL patient-derived cell lines (0.19 ± 0.01 compared to 1.0 ±0.02 at 1µM JQ1, p<0.0001).BRD4 has been shown to bind to di-acetylated TWIST1 in several cell types. TWIST1, a cellular regulatory protein often associated with epithelial-to-mesenchymal transition, is not normally expressed in CD4+ T-lymphocytes. Previous work from our lab has demonstrated aberrant overexpression of TWIST1 in CTCL patient CD4+ T-cells secondary to promoter hypomethylation. However, the role of TWIST1 expression in CTCL development has not been explored. We demonstrate that specific silencing of TWIST1 alone (0.51 ± 0.06, p=0.0046 vs 1.0 ± 0.054 for scrambled control), as well as in combination with silencing of BRD4 (0.51 ± 0.005, p=0.0013 vs scrambled control), results in significant decrease in miR-214 levels. Furthermore, we identify a novel target of miR-214, Kruppel-like factor 12 (KLF12), a protein with tumor suppressive functions in other cell types, but whose role in lymphoma is not known.Next, we performed in vivo treatment of IL-15 transgenic CTCL mice with 25mg/kg antagomiR-214 (n=4) or scrambled control (n=4) beginning at 8 weeks of age for 2 weeks. Scoring of the morphology and severity of skin lesions grossly and histologically demonstrated significant reduction in the extent of neoplastic disease in the skin of mice treated with antagomiR-214 (Figure 1B, C, mean ± SEM of expression in antagomir-214-treated mice vs vehicle-treated mice= 3.75 ± 0.239 vs 6.5 ± 0.144 out of a possible total score of 7, unpaired t-test, p=0.0027).We conclude that miR-214 overexpression in CTCL is in part driven by increased binding of TWIST1 and subsequent recruitment of BRD4 to the miR-214 promoter region. Specific targeting of miR-214 in vivo using an antagomiR results in reduced disease severity in a transgenic mouse model of CTCL. MiR-214 therefore represents a novel approach and a rational therapeutic target in CTCL. [Display omitted] DisclosuresPorcu:Tetralogic: Research Funding; Kiowa: Research Funding; Innate Pharma: Research Funding; Miragen: Research Funding; Galderma: Research Funding; Kura: Research Funding; Cell Medica: Research Funding; Celgene: Research Funding.

Epigenetics of cutaneous T-cell lymphoma: biomarkers and therapeutic potentials.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of skin-homing non-Hodgkin lymphomas. There are limited options for effective treatment of patients with advanced-stage CTCL, leading to a poor survival rate. Epigenetics plays a pivotal role in regulating gene expression without altering the DNA sequence. Epigenetic alterations are involved in virtually all key cancer-associated pathways and are fundamental to the genesis of cancer. In recent years, the epigenetic hallmarks of CTCL have been gradually elucidated and their potential values in the diagnosis, prognosis, and therapeutic intervention have been clarified. In this review, we summarize the current knowledge of the best-studied epigenetic modifications in CTCL, including DNA methylation, histone modifications, microRNAs, and chromatin remodelers. These epigenetic regulators are essential in the development of CTCL and provide new insights into the clinical treatments of this refractory disease.

16180 Clinical and histopathologic features of pityriasis lichenoides et varioliformis acuta in association with malignancy: An observational study

Background: Pityriasis lichenoides et varioliformis acuta (PLEVA) is a rare cutaneous inflammatory disorder of unknown etiology. Although PLEVA is generally accepted as a disease with a benign clinical course, rare reports of the development of cutaneous T-cell lymphoma exist. It is unknown whether PLEVA may be associated with other forms of malignancy. We report 10 cases of paraneoplastic PLEVA in association with extracutaneous malignancies.

The Involvement of STAT/SOCS Signaling in miRNA-Induced T Cell Exhaustion in Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell lymphoma (CTCL), collectively known as mycosis fungoides (MF) and Sézary syndrome (SS) arises from CD4+ T cells in a background of chronic inflammation. The chronic inflammation fosters the growth of CTCL cells and may facilitate T-cells exhaustion that is characterized by deprived effector function and sustained expression of inhibitory receptors. As a result, malignant CTCL cells escape immune surveillance and are not eliminated. At present, the roles of miRNAs or signaling pathways involved in the expression of immune checkpoints in CTCL has yet to be elucidated. Therefore, this project is aimed to elucidate how immune checkpoints are regulated by miRNAs, and how this regulation contributes to T-cell exhaustion and the development of CTCL. Methods: We first conducted miRNAseq analysis to assess the miRNA profile of 50 CTCL patient tumor samples. The sequencing data analysis was performed at the City of Hope Integrative Genomics Core. Next, we verified the expression of 3 highly upregulated miRNAs (miR-155, -21 and -130) from the miRNAseq analysis in 5 CTCL cell lines and tumor samples using qRT-PCR and in situ hybridization (ISH). Finally, we transfected the CTCL cell line Myla 2059 and HuT 78 with anti-miR-155, -21, -130 or Scramble (Scr) control using the Lonza nucleofection kit and nucleofector machine. Cell lysates were prepared 72 hours after transfection and then subjected to Western Blot analysis. We probed the blot with antibodies against SOCS, PTEN, pSTAT3 and GAPDH as a loading control. Results: The data analysis revealed that miR-155 had the highest correlation with CTLA-4 (r = 0.59, P &amp;lt; 0.0001), PD1 (r = 0.42, P = 0.0021), PD-L1 (r = 0.42, P = 0.0025), TIM3 (r = 0.63, P &amp;lt; 0.0001), LAG3 (r = 0.57, P &amp;lt; 0.0001), and ICOS (r = 0.74, P &amp;lt; 0.0001) mRNA; -21 had the highest correlation with PD-L1 (r = 0.44, P = 0.0012), TIM3 (r = 0.52, P &amp;lt; 0.0001), and ICOS (r = 0.37, P &amp;lt; 0.0073) mRNA; and -130 had the highest correlation with CTLA-4 (r = 0.5, P = 0.0002), PD-L1 (r = 0.54, P &amp;lt; 0.0001), TIM3 (r = 0.69, P &amp;lt; 0.0001), LAG3 (r = 0.49, P = 0.0003), and ICOS (r = 0.68, P &amp;lt; 0.0001) mRNA. qRT-PCR and ISH revealed that miRs-155, -21 and -130 were upregulated in all 5 CTCL cell lines and primary tumor samples. There was a dramatic increase in SOCS proteins and significant decrease in pSTAT3 expression in Myla 2059 and HuT 78 cells transfected with anti-miRs-155, -21 or -130, compared to cells transfected with Scr. Conclusions: Immune checkpoints and ligands like PD-L1 expression in CTCL are regulated by miRs-155, -21 and -130. The SOCS family of proteins, negative regulators of STAT signaling, are involved in miRs-155, -21 and -130-induced immune checkpoints expression in CTCL. Taken together, these results demonstrate the mechanisms of miRNA-induced T cell exhaustion and pave the way for the development of miRNA therapeutics in CTCL. Disclosures Rosen: Seattle Genetics: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy. Querfeld:Celgene: Research Funding; Stemline: Consultancy; Trillium: Consultancy; MiRagen: Consultancy; Kyowa Kirin: Consultancy; Bioniz: Consultancy; Helsinn: Consultancy.

Effect of expression of ICOS in cutaneous T-cell lymphoma and its targeting on killing of malignant cells.

e20040 Background: Advanced stage cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Immunomodulatory agents such as mogamulizumab, anti-KIR3DL2 and brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate (ADC) coupled to monomethyl-auristatin-E (MMAE), 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. 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 monoclonal antibodies with MMAE, in comparison to BV. We used ICOS+ CTCL cell lines (MyLa and MJ), murine xenograft models with MyLa and ICOS+ Patient Derived Xenografts (PDXs) from patients with SS and angioimmunoblastic T-cell lymphoma (AITL). 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 the 5 patients with node involvement. ICOS expression by circulating Sézary cells was strong: 69±7.3% versus 38.8±7.1% of non-tumoral CD4+ cells ( p&lt; 0.009; CI95%: 8.7-51.6); and 31±3.2% of CD4+ cells in HD ( p&lt; 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 ADCs. In a mouse xenograft model (MyLa), anti-ICOS ADCs provided a longer overall survival (OS) than BV (HR = 15.2;CI95%:3.2-71.1; p&lt; 0.0006). Finally, in ICOS+ PDXs anti-ICOS ADCs significantly improved OS, and reduced the number of tumor cells in the blood, bone marrow and spleen. No evidence of ADC toxicity was observed in treated mice. Conclusions: All together our results show that ICOS is a therapeutic target of interest in CTCLs and provide the preliminary basis for a therapeutic trial

YKL-40 Promotes Proliferation of Cutaneous T-Cell Lymphoma Tumor Cells through Extracellular Signal–Regulated Kinase Pathways

YKL-40, one of the chitinase-like proteins, is associated with the pathogenesis of a wide variety of human diseases through modulation of inflammation and tissue remodeling by its diverse roles in cell proliferation, differentiation, and survival. Emerging evidence shows that aberrantly expressed YKL-40 promotes the development of malignancies by inducing proliferation of tumor cells, cytokine production, and angiogenesis by acting on various stromal cells, immune cells, and tumor cells. In this study, we investigated the expression and function of YKL-40 in cutaneous T-cell lymphoma (CTCL). We first revealed that serum YKL-40 levels were increased in patients with CTCL and correlated with disease severity markers. We also found that YKL-40 was expressed by epidermal keratinocytes and tumor cells in lesional skin of CTCL by immunohistochemistry. Although YKL-40 did not affect cytokine production from CTCL cell lines, YKL-40 promoted the proliferation of Hut78 cells and HH cells invitro, which was dependent on extracellular signal-regulated kinase 1/2 pathways. Moreover, exogenous YKL-40 administration enhanced tumor growth of HH cells invivo. Our study has suggested that YKL-40 produced from epidermal keratinocytes and CTCL cells promoted the proliferation of CTCL cells through extracellular signal-regulated kinase 1/2 pathways in autocrine and paracrine manners, leading to development of CTCL.