Plasminogen Activating Inhibitor-1 Might Predict the Efficacy of Anti-PD1 Antibody in Advanced Melanoma Patients.

Plasminogen activating inhibitor-1 (PAI-1) plays crucial roles in the development of various cancers, including melanomas. Indeed, various pro-tumorigenic functions of PAI-1 in cancer progression and metastasis have been widely reported. Among them, PAI-1 is also reported as a key regulator of PD-L1 expression on melanoma cells through endocytosis, leading to abrogating the efficacy of anti-PD1 antibodies (Abs). These findings suggested that PAI-1 expression might predict the efficacy of anti-PD1 Abs. In this report, the expression and production of PAI-1 in melanoma patients were evaluated, and the immunomodulatory effects of PAI-1 on tumor-associated macrophages were investigated in vitro. Immunohistochemical staining of PAI-1 showed that PAI-1 expression on melanoma cells was significantly decreased in responders compared to non-responders. Moreover, baseline serum levels of PAI-1 were significantly decreased in responders compared to non-responders. Notably, PAI-1 decreased the production of various chemokines from monocyte-derived M2 macrophages in vitro, suggesting that PAI-1 might decrease tumor-infiltrating lymphocytes to hamper the anti-tumor effects of anti-PD1 Abs. These results suggest that baseline serum levels of PAI-1 may be useful as a biomarker for identifying patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.

Possible Roles of Proinflammatory Signaling in Keratinocytes Through Aryl Hydrocarbon Receptor Ligands for the Development of Squamous Cell Carcinoma.

Aryl hydrocarbon receptor (AhR) provides a deeper insight into the pathogenesis of cutaneous squamous cell carcinoma (cSCC). AhR ligands, such as 6-formylindolo[3,2-b] carbazole (FICZ), and 7,12-Dimethylbenz[a]anthracene (DMBA), constitute major substrates for the cytochrome P450 (CYP) family, and influence the expression of various cytokine genes, including IL-17 and IL-23-related genes via the AhR. On the other hand, proinflammatory cytokines could drive tumor progression through the TRAF-ERK5 signaling pathway in cSCC. From the above findings, we hypothesized that AhR ligands might enhance the mRNA expression of proinflammatory cytokines via the AhR, leading to the development of cSCC. The purpose of this study was to investigate (1) the immunomodulatory effects of FICZ and DMBA on normal human keratinocytes (NHKCs), focusing on IL-17, and related cytokines/chemokines (IL-23, IL-36γ, and CCL20), (2) the expression of these factors in AhR-dependent pathways using a two-stage chemically induced skin carcinogenesis mouse model, and (3) the expression of these factors in lesion-affected skin in cSCC. Both FICZ and DMBA augmented the expression of CYP1A1, p19, CCL20, and IL-36γ mRNA in NHKCs in vitro. Moreover, the mRNA expression of these proinflammatory factors, as well as IL-17, in mouse cSCC is significantly decreased in the AhR-(fl/fl) Krt5-(Cre) mice compared to wild type mice, leading to a decrease in the number of developed cSCC lesions. Furthermore, CCL20, IL-23, as well as IL-17, are detected in the lesion-affected skin of cSCC patients. Our study demonstrates a possible mechanism for the development of cSCC involving AhR-mediated signaling by epidermal keratinocytes and recruitment of Th17 cells.

Environmental pollutants and the immune response.

Environmental pollution is one of the most serious challenges to health in the modern world. Pollutants alter immune responses and can provoke immunotoxicity. In this Review, we summarize the major environmental pollutants that are attracting wide-ranging concern and the molecular basis underlying their effects on the immune system. Xenobiotic receptors, including the aryl hydrocarbon receptor (AHR), sense and respond to a subset of environmental pollutants by activating the expression of detoxification enzymes to protect the body. However, chronic activation of the AHR leads to immunotoxicity. KEAP1-NRF2 is another important system that protects the body against environmental pollutants. KEAP1 is a sensor protein that detects environmental pollutants, leading to activation of the transcription factor NRF2. NRF2 protects the body from immunotoxicity by inducing the expression of genes involved in detoxification, antioxidant and anti-inflammatory activities. Intervening in these sensor-response systems could protect the body from the devastating immunotoxicity that can be induced by environmental pollutants.

Aryl Hydrocarbon Receptor Modulates Carcinogenesis and Maintenance of Skin Cancers.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that responds to a wide range of chemicals, including chemical carcinogens such as dioxins and carcinogenic polyaromatic hydrocarbons, and induces a battery of genes associated with detoxification, proliferation, and immune regulation. Recent reports suggest that AHR plays an important role in carcinogenesis and maintenance of various types of skin cancers. Indeed, AHR is a susceptibility gene for squamous cell carcinoma and a prognostic factor for melanoma and Merkel cell carcinoma. In addition, the carcinogenic effects of ultraviolet (UV) and chemical carcinogens, both of which are major environmental carcinogenetic factors of skin, are at least partly mediated by AHR, which regulates UV-induced inflammation and apoptosis, the DNA repair system, and metabolic activation of chemical carcinogens. Furthermore, AHR modulates the efficacy of key therapeutic agents in melanoma. AHR activation induces the expression of resistance genes against the inhibitors of V600E mutated B-Raf proto-oncogene, serine/threonine kinase (BRAF) in melanoma and upregulation of programmed cell death protein 1 (PD-1) in tumor-infiltrating T cells surrounding melanoma. Taken together, these findings underscore the importance of AHR in the biology of skin cancers. Development of therapeutic agents that modulate AHR activity is a promising strategy to advance chemoprevention and chemotherapy for skin cancers.

Biomarkers for Predicting Efficacies of Anti-PD1 Antibodies.

Therapeutic options for treating advanced melanoma are progressing rapidly. Although anti-programmed cell death 1 (PD1) antibodies (e.g., nivolumab, pembrolizumab) have been approved as first-line and anchor drugs, respectively, for treating advanced melanoma, the efficacy appears limited as we expected, especially in Asian populations. Biomarkers to predict or evaluate the efficacy of anti-PD1 antibodies are needed to avoid subjecting patients to potentially severe adverse events associated with switching to other anti-melanoma drugs. This review focuses on the recent development of biomarkers for assessing the efficacy of anti-PD1 antibodies using routine blood tests such as the neutrophil-to-lymphocyte ratio, eosinophil ratio, serum markers such as lactate dehydrogenase, programmed cell death ligand 1 (PD-L1) expression on melanoma cells, microsatellite instability and mismatch repair deficiency assays, as well as soluble CD163, and tumor-associated macrophage-related chemokines (e.g., CXCL5, CXCL10).

Successful Treatment of Unresectable Advanced Melanoma by Administration of Nivolumab With Ipilimumab Before Primary Tumor Resection.

Ipilimumab, in combination with nivolumab, is one of the promising drugs that enhance the anti-tumor immune response of patients with advanced melanoma. Since the co-administration of nivolumab with ipilimumab in the neoadjuvant setting expands melanoma-reactive T cells at the primary site of melanoma and has a high rate of histological complete response, the pre-surgical administration of this combination could be the optimal therapy for unresectable advanced melanoma. In this report, a case of unresectable advanced melanoma treated successfully with administration of nivolumab with ipilimumab before primary tumor resection is presented. In addition, CD8+ T cells increased among the tumor-infiltrating lymphocytes that were surrounding melanoma cells and caspase 3+ cells. The present case suggests that pre-surgical administration of nivolumab with ipilimumab could be the optimal therapy for the treatment of unresectable advanced melanoma.

Malassezia-derived aryl hydrocarbon receptor ligands enhance the CCL20/Th17/soluble CD163 pathogenic axis in extra-mammary Paget's disease.

Malassezia yeast play a role in the pathogenesis of chronic dermatitis, especially in apocrine areas, by polarizing the local immunologic background to a Th2/Th17 state through aryl hydrocarbon receptor (AhR)-dependent pathways. Extra-mammary Paget's disease (EMPD) is an adenocarcinoma of apocrine origin, and except for cases associated with Malassezia yeast and their metabolites, the lesions typically develop in areas not exposed to environmental material. The purpose of this study was to investigate (a) the immunomodulatory effects of Malassezia metabolites on normal human keratinocytes (NHKCs), focusing on interleukin (IL)-17 and related cytokines/chemokines (IL-23, IL-36γ, CCL20), (b) the expression of these factors in lesion-affected skin in EMPD and (c) the activation of tumor-associated macrophages (TAMs) by these factors. Malassezia metabolites augmented the expression of cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), CCL20 and IL-36γ mRNA in NHKCs in vitro. In lesion-affected skin of patients with EMPD, epidermal keratinocytes expressed CYP1A1 and CCL20. In addition, Paget cells expressed CCL20 and IL-23. IL-17-producing cells were distributed adjacent to Paget cells. Compared to healthy donors, patients with EMPD exhibited significantly increased serum levels of soluble (s)CD163, CXCL5, CXCL10 and CCL20. In addition, serum levels of sCD163 decreased significantly following tumor resection. Our study demonstrates a possible mechanism for the development of EMPD involving AhR-mediated signalling by epidermal keratinocytes and RANKL-induced recruitment of Th17 cells and TAMs.

BRAF kinase inhibitors for treatment of melanoma: developments from early-stage animal studies to Phase II clinical trials.

INTRODUCTION: Approximately, 30.4-66.0% of cutaneous melanomas possess a mutation in the BRAF gene that activates downstream signaling through the mitogen-activated protein (MAP) kinase pathway; this provides an attractive target for the treatment of advanced melanoma. Although BRAF inhibitors rapidly suppress melanoma growth, median progression-free survival remains unsatisfactory. Recent clinical trials have investigated drugs that can optimally enhance and prolong the anti-melanoma effects of BRAF inhibitors. AREAS COVERED: This review discusses the development of BRAF inhibitor-based combination therapies for BRAF-mutant advanced melanoma. EXPERT OPINION: Future strategies for the treatment of advanced melanoma include novel combination therapies using BRAF/MEK inhibitors and immune checkpoints inhibitors or histone deacetylase inhibitors. These combination therapies might enhance antitumor responses against melanoma, prolonging survival in advanced melanoma patients. Further clinical studies are needed to optimize these novel combination therapies.

Serum Level of Soluble CD163 May Be a Predictive Marker of the Effectiveness of Nivolumab in Patients With Advanced Cutaneous Melanoma.

Antibodies against programmed cell death protein 1, such as nivolumab and pembrolizumab, are widely used for treating various cancers, including advanced melanoma. Nivolumab significantly prolongs survival in patients with metastatic melanoma, and sequential administration with lipilimumab may improve outcomes when switched at the appropriate time. Biomarkers are therefore needed to evaluate nivolumab efficacy soon after first administration. This study analyzed serum levels of soluble cluster of differentiation 163 (sCD163) in 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated using nivolumab. Serum levels of sCD163 were significantly increased after 6 weeks in responders compared to non-responders after initial administration of nivolumab for cutaneous melanoma. In contrast, no significant difference between responders and non-responders was seen among patients with non-cutaneous melanoma. These results suggest that sCD163 may be useful as a biomarker for selecting patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.

Serum levels of soluble CD163 and CXCL5 may be predictive markers for immune-related adverse events in patients with advanced melanoma treated with nivolumab: a pilot study.

Antibodies against PD-1, such as nivolumab and pembrolizumab, are widely used in the treatment of various cancers including advanced melanoma. The anti-PD-1 Ab significantly prolongs survival in patients with metastatic melanoma, and its administration in combination with local or systemic therapy may also lead to improved outcomes. Although anti-PD-1 Ab-based combined therapy might be effective for the treatment of advanced melanoma, the associated risk of irAEs is an important consideration. Therefore, being able to predict irAEs is of great interest to oncologists. The purpose of this study was to evaluate the value of using serum levels of sCD163 and CXCL5 to predict irAEs in patients with advanced melanoma who were administered nivolumab. To this end, we analyzed these serum levels in 46 cases of advanced melanoma treated with nivolumab. In addition, the tumor stroma was evaluated by immunohistochemistry and immunofluorescence. We measured the serum levels of sCD163 and CXCL5 on day 0 (immediately before nivolumab administration) and day 42. The serum absolute levels of sCD163 were significantly increased in patients who developed AEs (p = 0.0018). Although there was no significant difference in serum levels of CXCL5, the absolute value of CXCL5 could at least be a supportive marker for the increased absolute levels of serum sCD163. This study suggests that sCD163 and CXCL5 may serve as possible prognostic biomarkers for irAEs in patients with advanced melanoma treated with nivolumab.

HLA-DRB1*04:05 in two cases of Vogt-Koyanagi-Harada disease-like uveitis developing from an advanced melanoma patient treated by sequential administration of nivolumab and dabrafenib/trametinib therapy.

Although uveitis is reported as a rare adverse event (AE) associated with dabrafenib/trametinib therapy or nivolumab, the occurrence of severe uveitis is extremely rare. We describe two cases of Vogt-Koyanagi-Harada (VKH)-like uveitis developing after the sequential administration of nivolumab and dabrafenib/trametinib therapy. Interestingly, both cases had HLA-DRB1*04:05, which is strongly associated with VKH disease, and achieved biologically complete remission after the treatment for uveitis. Our cases suggest a possible correlation between VKH-like uveitis as an AE and the clinical outcomes of sequential administration of nivolumab and dabrafenib/trametinib therapy for the treatment of advanced melanoma.

Tumor-Associated Macrophages: Therapeutic Targets for Skin Cancer.

Tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) are significant components of the microenvironment of solid tumors in the majority of cancers. TAMs sequentially develop from monocytes into functional macrophages. In each differentiation stage, TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment (e.g., expression of immune checkpoint molecules, production of Treg-related chemokines and cytokines, production of arginase I). Although the main population of TAMs is immunosuppressive M2 macrophages, TAMs can be modulated into M1-type macrophages in each differential stage, leading to the suppression of tumor growth. Because the administration of certain drugs or stromal factors can stimulate TAMs to produce specific chemokines, leading to the recruitment of various tumor-infiltrating lymphocytes, TAMs can serve as targets for cancer immunotherapy. In this review, we discuss the differentiation, activation, and immunosuppressive function of TAMs, as well as their benefits in cancer immunotherapy.

The Expression of Matrix Metalloproteinases in Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)-expressing Cancer of Apocrine Origin.

Primary cutaneous apocrine carcinoma (PCAC) is a rare and highly aggressive tumor entity. Since there is no conventional therapy for advanced PCAC, exploratory treatments are sometimes used. As we previously reported, receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL)/RANK signaling on M2 macrophages promotes the production of chemokines and proinflammatory cytokines to maintain the immunosuppressive tumor environment of extramammary Paget's disease (EMPD). Since EMPD is a skin adenocarcinoma of apocrine gland origin that expresses high levels of RANKL and matrix metalloproteinase (MMP) 7, and EMPD is associated with the presence of RANK+ M2 macrophages, we hypothesized that tumor-associated macrophages (TAMs) in adenocarcinomas such as PCAC might also express RANKL and MMP7. MATERIALS AND METHODS: We employed immunohistochemical staining of RANKL and MMP7 in the lesional skin from five patients with PCAC, and microarray analysis of MMPs using human monocyte-derived macrophages. RESULTS: According to DNA microarray analysis, the expression of MMP1 and MMP25 was augmented. The DNA microarray results were verified by using real-time polymerase chain reaction (RT-PCR). Immunohistochemical staining of MMP1 and MMP25 as well as chemokine (C-C motif) ligand (CCL) 5 in the lesional skin from five patients with PCAC showed a substantial number of MMP1-bearing cells and MMP25-bearing cells, as well as CCL5-producing cells, that were distributed in the lesional skin. CONCLUSION: Our study suggests that the RANKL/RANK pathway contributes to the development and maintenance of the immunosuppressive tumor microenvironment and denosumab may be a promising adjuvant therapy targeting TAMs in cancer of apocrine origin.

Phase I study of nivolumab combined with IFN-ß for patients with advanced melanoma.

The efficacy of nivolumab is greater than that of other anti-melanoma drugs, so nivolumab-based combined therapies that enhance anti-tumor immune responses in patients with metastatic melanoma are of great interest to dermato-oncologists. As we have previously reported, IFN-ß enhances the anti-tumor immune response of anti-PD-1 antibodies against B16F10 melanoma in vivo. To explore the potential of this property of IFN-ß as part of a combination therapy for the treatment of metastatic melanoma patients, we performed a phase 1 trial, using a traditional rule-based 3 + 3 design, on patients with advanced melanoma. The nivolumab dose was fixed at 2 mg/kg, every 3 weeks. IFN-ß was administered to three groups at doses of 1 million, 2 million, and 3 million units, respectively. Dose-limiting toxicities were defined as any grade 3-5 adverse events occurring between day 0 and day 42 that might possibly be related to nivolumab and IFN-ß. Of the nine patients who received this combined therapy, none experienced dose-limiting toxicities, and all completed the treatment phase of the study. Patient follow-up continued for 6 months following the final treatment. There were two complete responses (22%) and one partial response (11%), all of which occurred in patients who had received monthly IFN-ß immediately prior to the study. In this study, we determined the safe dose of IFN-ß, when combined with nivolumab, to be 3 million units. To determine the efficacy of this combination therapy, further phase II trials are required.

Immunomodulatory Effect of Imiquimod Through CCL22 Produced by Tumor-associated Macrophages in B16F10 Melanomas.

BACKGROUND/AIM: Tumor-associated macrophages (TAMs), together with splenic CD11b+ cells, help maintain the tumor microenvironment. The immunomodulatory compound imiquimod (IQM) stimulates innate immune cells, including macrophages, to induce antitumor effects. In order to elucidate the effects of IQM on the tumor microenvironment, we investigated the immunomodulatory effect of IQM during melanoma growth by using the B16F10 melanoma model. MATERIALS AND METHODS: To elucidate the immunomodulatory effects of IQM on the tumor microenvironment, we isolated CD11b+ TAMs and splenic CD11b+ cells and evaluated the immunomodulatory effects of IQM, using the B16F10 melanoma model. RESULTS: IQM suppressed B16F10 melanoma growth in parallel with reduction of Foxp3+ regulatory T cells (Tregs) at the tumor site, caused by the down-regulation of CCL22 production by tumor-derived and splenic CD11b+ cells. Subsequently, we investigated the antitumor or tumor-loading effects of splenic CD11b+ cells on B16F10 melanoma growth in vivo. B16F10 melanoma growth was accelerated by splenic CD11b+ cells from untreated mice, but was inhibited by splenic CD11b+ cells from IQM-treated mice. Consistent with these results, Foxp3+ Tregs were significantly decreased in tumors of mice implanted with both melanoma and splenic CD11b+ cells from topical IQM-treated mice. Furthermore, intratumoral administration of anti-CCL22 antibody inhibited B16F10 melanoma growth by decreasing Treg recruitment at the tumor site. CONCLUSION: Our results suggest a possible mechanism for the antitumor immune response induced by IQM through tumor-associated macrophages.