High-Risk Non-Melanoma Skin Cancers: Biological and Therapeutic Advances.

Nonmelanoma skin cancers (NMSCs) are the most common cancers, with high-risk NMSCs sharing features such as poor histologic differentiation, invasion into deeper layers, and anatomic location. NMSC includes basal cell carcinoma, cutaneous squamous cell carcinoma, and Merkel cell carcinoma. Herein, the authors describe advances in understanding the genetic mechanisms of malignant transformation and the composition of tumor microenvironment for these cancers. They summarize recent therapeutic advances, including targeted therapy and immunotherapy for NMSCs. Effective skin protection against ultraviolet radiation-induced carcinogenesis remains an urgent unmet need for NMSC prevention. The authors highlight immune-based interventions as novel strategies to address this need.

Cytotoxic CD4+ T cells eliminate senescent cells by targeting cytomegalovirus antigen.

Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases, including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old skin compared with young skin. However, they did not increase with advancing age in the elderly. Increased CXCL9 and cytotoxic CD4+ T cells (CD4 CTLs) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTLs eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner, and HCMV-gB activated CD4 CTLs from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which CD4 CTLs can directly eliminate through the recognition of the HCMV-gB antigen.

Combined PD-1, BRAF and MEK inhibition in BRAFV600E colorectal cancer: a phase 2 trial.

While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and lack durability. Preclinical data suggest that BRAF/MAPK pathway inhibition may augment the tumor immune response. We performed a proof-of-concept single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC. The primary end point was overall response rate, and the secondary end points were progression-free survival, disease control rate, duration of response and overall survival. The study met its primary end point with a confirmed response rate (24.3% in all patients; 25% in microsatellite stable patients) and durability that were favorable relative to historical controls of BRAF-targeted combinations alone. Single-cell RNA sequencing of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed greater induction of tumor cell-intrinsic immune programs and more complete MAPK inhibition in patients with better clinical outcome. Immune program induction in matched patient-derived organoids correlated with the degree of MAPK inhibition. These data suggest a potential tumor cell-intrinsic mechanism of cooperativity between MAPK inhibition and immune response, warranting further clinical evaluation of optimized targeted and immune combinations in CRC. ClinicalTrials.gov registration: NCT03668431.

Increased expression of squamous cell carcinoma antigen 1 and 2 in mycosis fungoides and Sézary syndrome

Background: Squamous cell carcinoma antigen (SCCA) was originally isolated as tumour-specific antigens in uterine cervix carcinoma. These comprise two similar proteins, SCCA1 and SCCA2, and both are induced by type 2 cytokines such as interleukin (IL)-4 and IL-13. The involvement of these antigens in atopic dermatitis has been reported, however, the role in mycosis fungoides (MF) and Sézary syndrome (SS), which are also linked with type 2 cytokines, remains to be seen. Objectives: This study investigated a possible association between SCCA1/2 and MF/SS. Materials & Methods: We compared serum levels of SCCA1/2 between MF/SS patients and healthy controls. We also examined the correlation between serum SCCA1/2 levels in MF/SS patients and clinical disease markers. The expression of SCCA1/2 in skin samples was examined by immunohistochemistry. Results: The serum levels of SCCA1/2 in MF/SS patients were significantly higher than those in normal controls and correlated with clinical disease markers. Immunohistochemical staining showed upregulated expression of SCCA1/2 in MF/SS lesional skin. Conclusion: Enhanced SCCA1/2 expression may contribute to the progression of MF/SS. Measurement of serum SCCA1/2 levels may become a useful tool to evaluate the progression or therapeutic effects of MF/SS.

Alarmin Cytokines as Central Regulators of Cutaneous Immunity.

Skin acts as the primary interface between the body and the environment. The skin immune system is composed of a complex network of immune cells and factors that provide the first line of defense against microbial pathogens and environmental insults. Alarmin cytokines mediate an intricate intercellular communication between keratinocytes and immune cells to regulate cutaneous immune responses. Proper functions of the type 2 alarmin cytokines, thymic stromal lymphopoietin (TSLP), interleukin (IL)-25, and IL-33, are paramount to the maintenance of skin homeostasis, and their dysregulation is commonly associated with allergic inflammation. In this review, we discuss recent findings on the complex regulatory network of type 2 alarmin cytokines that control skin immunity and highlight the mechanisms by which these cytokines regulate skin immune responses in host defense, chronic inflammation, and cancer.

CD147-Cyclophilin a Interactions Promote Proliferation and Survival of Cutaneous T-Cell Lymphoma.

CD147, a transmembrane glycoprotein that belongs to the immunoglobulin superfamily, and cyclophilin A (CypA), one of the binding partners of CD147, are overexpressed in tumor cells and associated with the progression of several malignancies, including both solid and hematological malignancies. However, CD147 and CypA involvement in cutaneous T-cell lymphoma (CTCL) has not been reported. In this study, we examined CD147 and CypA expression and function using clinical samples of mycosis fungoides (MF) and Sézary syndrome (SS) and CTCL cell lines. CD147 and CypA were overexpressed by tumor cells of MF/SS, and CypA was also expressed by epidermal keratinocytes in MF/SS lesional skin. Serum CypA levels were increased and correlated with disease severity markers in MF/SS patients. Anti-CD147 antibody and/or anti-CypA antibody suppressed the proliferation of CTCL cell lines, both in vitro and in vivo, via downregulation of phosphorylated extracellular-regulated kinase 1/2 and Akt. These results suggest that CD147-CypA interactions can contribute to the proliferation of MF/SS tumor cells in both a autocrine and paracrine manner, and that the disruption of CD147-CypA interactions could be a new therapeutic strategy for the treatment of MF/SS.

Increased Regulatory T Cells and Decreased Myeloid-Derived Suppressor Cells Induced by High CCL17 Levels May Account for Normal Incidence of Cancers among Patients with Atopic Dermatitis.

The incidence of cancers in atopic dermatitis (AD) is not increased, although the Th2-dominant environment is known to downregulate tumor immunity. To gain mechanistic insights regarding tumor immunity in AD, we utilized CCL17 transgenic (TG) mice overexpressing CCL17, which is a key chemokine in AD. Tumor formation and lung metastasis were accelerated in CCL17 TG mice when melanoma cells were injected subcutaneously or intravenously. Flow cytometric analysis showed increases in regulatory T cells (Tregs) in lymph nodes in CCL17 TG mice with high mRNA levels of IL-10 and Foxp3 in tumors, suggesting that Tregs attenuated tumor immunity. The frequency of myeloid-derived suppressor cells (MDSCs), however, was significantly decreased in tumors of CCL17 TG mice, suggesting that decreased MDSCs might promote tumor immunity. Expression of CXCL17, a chemoattractant of MDSCs, was decreased in tumors of CCL17 TG mice. Depletion of Tregs by the anti-CD25 antibody markedly reduced tumor volumes in CCL17 TG mice, suggesting that tumor immunity was accelerated by the decrease in MDSCs in the absence of Tregs. Thus, CCL17 attenuates tumor immunity by increasing Tregs and Th2 cells, while it decreases MDSCs through reductions in CXCL17, which may work as a "safety-net" to reduce the risk of malignant tumors in the Th2-dominant environment.

Mycosis fungoides and Sézary syndrome tumor cells express epidermal fatty acid-binding protein, whose expression decreases with loss of epidermotropism.

Fatty acid binding protein (FABP) is a family of transport proteins for fatty acid (FA). Epidermal FABP (E-FABP) is highly expressed by resident memory T cells (TRM ) in the skin. It supports the uptake of exogenous FA for long-term survival of skin TRM . Mycosis fungoides (MF) is regarded as malignancy of skin TRM . In this study, we investigated E-FABP expression in psoriasis vulgaris (PV), atopic dermatitis (AD), MF, and Sézary syndrome (SS). E-FABP mRNA levels in PV were much higher than those in healthy controls. E-FABP mRNA levels in AD and MF/SS lesional skin were also significantly higher than those of normal skin. By immunohistochemical staining, E-FABP was positive in MF/SS lesional skin. Interestingly, E-FABP was stained positive in epidermotropic lymphoid cells in patch, plaque, and erythrodermic lesions of MF/SS, suggesting that a part of tumor cells expressed E-FABP. In tumorous lesions, however, most dermal tumor cells were negative for E-FABP. Immunohistochemical staining using patch/plaque lesions and tumorous lesions from the same patients also revealed that E-FABP expression decreased in tumorous lesions. Our study has suggested that MF/SS tumor cells express E-FABP, whose expression decreases with loss of epidermotropism.

Unusual type of scleromyxedema with multiple subcutaneous nodules, IgM-λ paraproteinemia, and scleroderma-like microangiopathy.

Scleromyxedema is a mysterious cutaneous mucinosis of unknown etiology. Various types of scleromyxedema variant have been reported, which often give us a clue to understand the key aspects of this disease. Here, we describe a woman with highly unusual type of scleromyxedema. In addition to the rare manifestations of multiple subcutaneous nodules and IgM-λ paraproteinemia, our patient showed several characteristic symptoms of scleroderma such as shortened nails and fingertips, sclerodactyly, and bone resorption of fingertips and mandibles as a result of peripheral circulatory insufficiency, although this disease is known to be pathophysiologically different from scleroderma. A skin biopsy revealed cutaneous microvascular stenosis and occlusion due to intravascular mucin deposition and fibrotic changes, suggesting that scleromyxedema potentially develops peripheral circulatory disorders and other vascular involvement. The subcutaneous nodules were responsive to high-dose intravenous immunoglobulin. Scleromyxedema can represent a wide variety of systemic involvement, and therefore, we should pay attention to those symptoms as well as skin lesions.

Increased Expression of Delta-like Ligand 4 in Mycosis Fungoides.

In many malignancies, dysregulation of the Notch pathways, composed of 4 Notch receptors (Notch1-4) and 5 Notch ligands (Jagged1-2, Delta-like ligand-1, 3-4), is associated with their development. In mycosis fungoides, interaction between Notch1 and Jagged1 is known to activate the Notch pathways and promote the proliferation of tumour cells. However, the involvement of other Notch ligands has not been reported. This study investigated the roles of Delta-like ligand 4 in mycosis fungoides. Delta-like ligand 4 mRNA levels in lesional skin of patients with mycosis fungoides were significantly elevated compared with those of normal controls, and correlated with disease-specific mortality. Immunohistochemical staining demonstrated prominent expression of Delta-like ligand 4 on vascular endothelial cells and tumour cells in mycosis fungoides lesional skin. In addition, Delta-like ligand 4 augmented the proliferation of cutaneous T-cell lym-phoma cell lines. These results suggest that enhanced Delta-like ligand 4 expression may contribute directly to the progression of mycosis fungoides through proliferating tumour cells.

Primary cutaneous γδ T-cell lymphoma with unusual immunophenotype: A case report and review of published work.

Primary cutaneous γδ T-cell lymphoma (CGD-TCL) is a rare form of primary cutaneous lymphoma. The histopathological features of CGD-TCL are still unclear because of its rarity. Here, we report a case of a 77-year-old Japanese man who presented with a 9-month history of erythematous plaques on his left forearm. Skin biopsy specimens revealed the infiltration of atypical medium/large-sized lymphocytes from the epidermis to the deep dermis. Atypical lymphocytes were positive for CD3, CD5, CD8 and Vδ1, and negative for CD4, CD7, CD56, EBER-ISH, intracellular antigen-1, granzyme B and perforin. CD30 was partially expressed. We also reviewed 246 cases of CGD-TCL from the published work. CD4- CD8- double-negative cases were 113 of 196 cases (57.6%), followed by CD4- CD8+ cases (52/196, 26.5%). CD5 was expressed in 25.8% of the cases (34/132). At least one cytotoxic molecule marker was expressed in 150 of 160 cases (93.8%). Some cases showed an indolent clinical course, especially in mycosis fungoides-like CGD-TCL cases. CD5 positivity and lack of cytotoxic molecule expression could be associated with a better prognosis. In addition, CD30 expression was found in approximately half of CGD-TCL cases (51/112 cases), suggesting that brentuximab vedotin could be a good treatment option for such patients. Further studies with more cases with detailed clinical and pathological information are necessary to elucidate the etiology and prognostic markers of this entity.

Increased IL-26 Expression Promotes T Helper Type 17- and T Helper Type 2-Associated Cytokine Production by Keratinocytes in Atopic Dermatitis.

Whereas atopic dermatitis (AD) is considered as a T helper 2 (Th2)-centered disease, IL-17-producing Th (Th17) cells are also activated in AD lesional skin. However, the relationship between Th17 responses and Th2 responses in AD is still to be elucidated. Although Th17 cells are increased in AD skin, the expression and function of IL-26, which is also produced by Th17 cells, in AD are still unknown. In this report, we demonstrated that IL-26 mRNA expression levels were elevated in AD lesional skin compared with healthy controls and that IL-26-producing cells were increased in AD lesional skin by immunohistochemistry. Furthermore, IL-26 promoted IL-8, IL-1ß, chemokine (C-C motif) ligand 20, IL-33, and ß-defensin 2 production in keratinocytes through phosphorylation of signal transducer and activator of transcription 1 and signal transducer and activator of transcription 3. Selective JAK inhibitors for JAK1, JAK2, and tyrosine kinase 2 blocked IL-26-induced cytokine production in keratinocytes. We also showed that injection of IL-26 exacerbated an oxazolone-induced AD mouse model and upregulated Th2 and Th17 cytokine expression in vivo. Because previous studies indicate that the above molecules induced by IL-26 can promote Th17 and/or Th2 immune responses, IL-26 may play an important role for bridging between Th17 and Th2 responses, resulting in the development of AD.

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 in vitro, which was dependent on extracellular signal-regulated kinase 1/2 pathways. Moreover, exogenous YKL-40 administration enhanced tumor growth of HH cells in vivo. 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.

Thrombospondin-1 promotes tumor progression in cutaneous T-cell lymphoma via CD47.

CD47 is highly expressed on various hematopoietic malignancies, and enables cancer cells to avoid immunosurveillance. Its ligand, thromobospondin-1 (TSP-1) is a multifunctional protein, and CD47/TSP-1 interactions promote tumor progression in various malignancies. In this study, we investigated roles of TSP-1 and CD47 in cutaneous T-cell lymphoma (CTCL). Flow cytometric analysis and immunohistochemistry showed that CTCL tumor cells and CTCL cell lines (Hut78, HH, and MyLa cells) overexpressed CD47 compared with normal CD4+ T cells. Overexpression of CD47 was partially induced by high c-Myc expression in CTCL tumor cells. TSP-1 mRNA expression levels in CTCL lesional skin were higher than those in normal skin and correlated with increased risk of disease-related death. Moreover, TSP-1 was expressed on CTCL tumor cells by immunohistochemistry. Serum soluble TSP-1 levels in patients with Sézary syndrome were significantly elevated. TSP-1 promotes proliferation and survival of CTCL tumor cells, which is inhibited by anti-CD47 neutralizing antibody or CD47 knockdown. Stimulation with TSP-1 also induces cell migration and in vivo growth. These effects were mediated by phosphorylation of ERK1/2 and AKT and expression of survivin. Collectively, our findings prompt a novel therapeutic approach to CTCL based on discovery that CD47/TSP-1 interactions play important roles in progression of CTCL.

Possible therapeutic applicability of galectin-9 in cutaneous T-cell lymphoma.

BACKGROUND: Galectin-9, a member of the galectin family, can promote tumor growth through inducing apoptosis in anti-tumor immune cells via T cell immunoglobulin and mucin domain 3 (TIM-3). On the other hand, galectin-9 also induces tumor cell apoptosis in many malignancies and thought to have potential as an anti-cancer agent. OBJECTIVE: To examine the expression and therapeutic applicability of galectin-9 in cutaneous T-cell lymphoma (CTCL). METHODS: Galectin-9 expression in lesional skin and sera was measured using CTCL samples. The effect of galectin-9 on CTCL cell lines was investigated in vitro. We also examined effect of galectin-9 on tumor growth of CTCL cells in immune-deficient mice. Moreover, we examined the efficacy of galectin-9, anti-TIM-3 blocking antibody, or their combination on tumor growth of EL-4 cells in wild-type mice. RESULTS: Galectin-9 was expressed on tumor cells in lesional skin of CTCL and the expression levels were associated with decreased CD8+ T-cell infiltration. Serum galectin-9 levels were correlated with disease severity markers. High-dose galectin-9 induced cell death of CTCL cell lines through activation of caspase-3 and caspase-9, independently of TIM-3. High-dose galectin-9 suppressed the growth of CTCL cells and EL-4 cells in vivo. Furthermore, additional anti-TIM-3 blocking antibody administration to galectin-9 achieved greater inhibition of tumor growth compared to single administration. CONCLUSION: Galectin-9 expression on tumor cells may be associated with CTCL progression through attenuating anti-tumor immunity. On the other hand, exogenous high-dose galectin-9 administration can be a therapeutic strategy for CTCL and anti-TIM-3 blocking antibody can augment the efficacy of galectin-9.

Novel and Future Therapeutic Drugs for Advanced Mycosis Fungoides and Sézary Syndrome.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphoma. The majority of MF cases present with only patches and plaques and the lesions are usually limited to the skin. On the other hand, in some cases, patients show skin tumors or erythroderma followed by lymph node involvement and rarely visceral organ involvement. SS is a rare, aggressive cutaneous T-cell lymphoma marked by exfoliative erythroderma, lymphadenopathy, and leukemic blood involvement. Because patients with relapsed or refractory MF/SS display a poor prognosis and the current treatment options are characterized by high rates of relapse, there is unmet need for the efficient treatment. This review provides a discussion of the recent and future promising therapeutic approaches in the management of advanced MF/SS. These include mogamulizumab, brentuximab vedotin, alemtuzumab, immune checkpoint inhibitors, IPH4102 (anti-KIR3DL2 antibody), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, belinostat, and resminostat), pralatrexate, forodesine, denileukin diftitox, duvelisib, lenalidomide, and everolimus.

Expression of CCR3 and CCR4 Suggests a Poor Prognosis in Mycosis Fungoides and Sézary Syndrome.

Tumor cells in cutaneous T-cell lymphoma express limited numbers of chemokine receptors. We investigated the expression patterns of CXCR3, CCR3, CCR4 and CCR10 in mycosis fungoides, Sézary syndrome, lym-phomatoid papulosis and anaplastic large cell lymphoma in 121 skin biopsy samples. CXCR3 was expressed in 86% of mycosis fungoides cases but in no anaplastic large cell lymphoma cases. CCR3 was expressed in 73% of cases of CD30+ lymphoproliferative disorders such as lymphomatoid papulosis and anaplastic large cell lymphoma. Mycosis fungoides/Sézary syndrome patients with high CCR3 or CCR4 expression had a poorer survival prognosis than mycosis fungoides/Sézary syndrome patients whose tumor cells did not express these receptors. CCR10 was expressed in 50% of mycosis fungoides/Sézary syndrome cases and in 13% of cases with CD30+ lym-phoproliferative disorders. These results suggest that differential patterns of CXCR3, CCR3, CCR4 and CCR10 expression are useful for the diagnosis of cutaneous T-cell lymphoma. Moreover, expression of CCR3 or CCR4 suggests a poor prognosis in mycosis fungoides/Sézary syndrome.

Keratinocyte Proline-Rich Protein Deficiency in Atopic Dermatitis Leads to Barrier Disruption.

Atopic dermatitis is a common inflammatory skin disease caused by the interaction of genetic and environmental factors. By allelic copy number analysis at missense single-nucleotide polymorphisms on 26 genes with copy number variation, we identified a significant association between atopic dermatitis and human KPRP. Human KPRP expression, which was localized to the upper granular layer of epidermis, was significantly decreased in atopic dermatitis compared with normal skin. KPRP was histologically colocalized with loricrin and was mainly detected in cytoskeleton fractions of human keratinocytes. To further investigate the role of KPRP in skin, Kprp-knockout mice were generated. Heterozygous knockout (Kprp+/-) mice exhibited reduced KPRP expression to level a similar to that of human AD lesional skin. Kprp+/- mice showed abnormal desmosome structure and detachment of lower layers of the stratum corneum. Percutaneous inflammation by topical application of croton oil or oxazolone was enhanced, and epicutaneous immunization with ovalbumin induced a high level of IgE in Kprp+/- mice. Our study, started from allelic copy number analysis in human AD, identified the importance of KPRP, the decrease of which leads to barrier dysfunction.