Effects of temozolomide on tumor mutation burden and microsatellite instability in melanoma cells.

The efficacy of combination therapy with an immune checkpoint inhibitor (ICI) and cytotoxic chemotherapeutic agents has been investigated in cancer, including melanoma. Before ICIs were introduced, dacarbazine or temozolomide (TMZ) were used to treat melanoma. Several studies using glioma or colorectal cancer cells showed that TMZ can increase the tumor mutation burden (TMB) and induce mismatch repair (MMR) deficiency associated with microsatellite instability (MSI). These could increase immunoreactivity to an ICI, but this has not been evaluated in melanoma cells. We investigated the effects of TMZ on MSI status and TMB in melanoma cells. To evaluate the TMB, we performed whole-exome sequencing using genomic DNA from the human melanoma cell lines Mel18, A375, WM266-4, G361, and TXM18 before and after TMZ treatment. Polymerase chain reaction amplification of five mononucleotide repeat markers, BAT25, BAT26, NR21, NR24, and MONO27, was performed, and we analyzed changes in the MSI status. In all cell lines, the TMB was increased after TMZ treatment (the change amount of TMB with ≤ 5% variant allele frequency [VAF] was 18.0-38.3 mutations per megabase) even in the condition without obvious cytological damage. MSI after TMZ treatment was not observed in any cells. TMZ increased TMB but did not change MSI status in melanoma cells.

Molecular Events in the Melanogenesis Cascade as Novel Melanoma-Targeted Small Molecules: Principle and Development.

Malignant melanoma is one of the most malignant of all cancers. Melanoma occurs at the epidermo-dermal interface of the skin and mucosa, where small vessels and lymphatics are abundant. Consequently, from the onset of the disease, melanoma easily metastasizes to other organs throughout the body via lymphatic and blood circulation. At present, the most effective treatment method is surgical resection, and other attempted methods, such as chemotherapy, radiotherapy, immunotherapy, targeted therapy, and gene therapy, have not yet produced sufficient results. Since melanogenesis is a unique biochemical pathway that functions only in melanocytes and their neoplastic counterparts, melanoma cells, the development of drugs that target melanogenesis is a promising area of research. Melanin consists of small-molecule derivatives that are always synthesized by melanoma cells. Amelanosis reflects the macroscopic visibility of color changes (hypomelanosis). Under microscopy, melanin pigments and their precursors are present in amelanotic melanoma cells. Tumors can be easily targeted by small molecules that chemically mimic melanogenic substrates. In addition, small-molecule melanin metabolites are toxic to melanocytes and melanoma cells and can kill them. This review describes our development of chemo-thermo-immunotherapy based on the synthesis of melanogenesis-based small-molecule derivatives and conjugation to magnetite nanoparticles. We also introduce the other melanogenesis-related chemotherapy and thermal medicine approaches and discuss currently introduced targeted therapies with immune checkpoint inhibitors for unresectable/metastatic melanoma.

Functional Interplay between IL-9 and Peptide YY Contributes to Chronic Skin Inflammation.

Complex interactions between keratinocytes and various cell types, such as inflammatory cells and stromal cells, contribute to the pathogenesis of chronic inflammatory skin lesions. In proinflammatory cytokine‒mediated disease settings, IL-9 plays a pathological role in inflammatory dermatitis. However, IL-9‒related mechanisms remain incompletely understood. In this study, we established tamoxifen-induced keratinocyte-specific IL-9RA-deficient mice (K14CRE/ERTIl9raΔ/Δ mice) to examine the role of IL-9 in multicellular interactions under chronic skin inflammatory conditions. Studies using an imiquimod-induced psoriasis-like model showed that K14CRE/ERTIl9raΔ/Δ mice exhibited a significantly reduced severity of dermatitis and mast cell infiltration compared with control K14WTIl9rafl/fl mice. Transcriptome analyses of psoriasis-like lesions showed that the level of peptide Y-Y (Pyy), a member of the neuropeptide Y family, was markedly downregulated in K14CRE/ERTIl9raΔ/Δ epidermis. Pyy blockade suppressed epidermal thickening and mast cell numbers in imiquimod-treated wild-type mice. Together with in vitro studies indicating that Pyy induced IL-9 production and chemotactic activity in bone marrow‒derived mast cells, these findings suggest that Pyy-mediated interplay between keratinocytes and mast cells contributes to psoriasiform inflammation. Further investigation focusing on the IL-9‒Pyy axis may provide valuable information for the development of new treatment modalities for inflammatory dermatitis.

Difference in immunohistochemical findings among anti-PD-L1 antibodies and their relationships with CD4+ and CD8+ T cells in Japanese melanoma patients.

BACKGROUND: The immunohistochemical evaluation of programmed death ligand 1 (PD-L1) is important for selecting treatments. Several antibodies are available for such evaluations, but data regarding the differences in the antibodies' positivity are limited in melanoma, particularly the acral and mucosal types. We investigated the differences in melanoma tissues' PD-L1 expression among the commonly used PD-L1 antibodies and then evaluated the relationship between PD-L1+ tumor cells and tumor-infiltrating lymphocytes (TILs). PATIENTS AND METHODS: We examined 56 primary lesions and 8 metastatic lymph node samples from 56 Japanese patients with melanoma (28 acral melanoma, 8 mucosal melanoma, 18 cutaneous melanoma, 2 unknown). Immunohistochemical staining was performed using three primary antibodies against PD-L1 (E1L3N, SP142, and 28-8). PD-L1-positive staining in tumor cells was defined as ≥ 1% expression. RESULTS: The positive rates were 25.0% for 28-8, 34.0% for E1L3N, and 34.0% for SP142 in 64 samples. The positive rates of acral melanoma were 10.7% for 28-8, 21.4% for E1L3N, and 21.4% for SP142. The positive rate of mucosal melanoma for which all three antibodies reacted was 12.5%. The positive rates of cutaneous melanoma were 55.6% for 28-8, 66.7% for E1L3N, and 66.7% for SP142. Significant relationships were observed among the PD-L1+ tumor cells, CD4+ TILs, and CD8+ TILs (p < 0.001). CONCLUSION: The staining results by E1L3N, SP142, and 28-8 antibodies were within the allowable range, although the positive rates by E1L3N and P142 were slightly higher than that of 28-8. CD4+ TILs and CD8+ TILs were quantitatively correlated with PD-L1-positive tumor cells.

Immunomodulation of Melanoma by Chemo-Thermo-Immunotherapy Using Conjugates of Melanogenesis Substrate NPrCAP and Magnetite Nanoparticles: A Review.

A major advance in drug discovery and targeted therapy directed at cancer cells may be achieved by the exploitation and immunomodulation of their unique biological properties. This review summarizes our efforts to develop novel chemo-thermo-immunotherapy (CTI therapy) by conjugating a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine), with magnetite nanoparticles (MNP). In our approach, NPrCAP provides a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It also functions as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Moreover, the utilization of MNP is a platform to develop thermo-immunotherapy because of heat shock protein (HSP) expression upon heat generation in MNP by exposure to an alternating magnetic field (AMF). This comprehensive review covers experimental in vivo and in vitro mouse melanoma models and preliminary clinical trials with a limited number of advanced melanoma patients. We also discuss the future directions of CTI therapy.

Genetic analyses of a secondary poroma and trichoblastoma in a HRAS-mutated sebaceous nevus.

A sebaceous nevus is a congenital skin hamartoma caused by postzygotic HRAS or KRAS mosaic mutations. With age, affected individuals may develop secondary tumors within a sebaceous nevus. RAS mutations are harbored from the onset of sebaceous nevus, and further mutations can be expected to be required in order to explain the initiation of secondary tumors. However, genetic analyses of the secondary tumors have not been conducted. Herein, we describe the rare coexistence of a poroma and a trichoblastoma arising in a sebaceous nevus. This is the first report of an investigation of multiple genes in a secondary tumor in an SN. First, HRAS c.37G>C, which is the common mutation in sebaceous nevus, was detected in all three lesions (sebaceous nevus, poroma, and trichoblastoma). Next, to elucidate the potential second-hit mutations in the secondary poroma and trichoblastoma, we applied a panel sequencing for skin cancers that was newly developed in our institution. Our comparison of the mutational profile of 95 skin cancer-related genes in each of the three lesions newly revealed TP53 p.R158P in the poroma and NOTCH2 p.G329S in the trichoblastoma. TP53 p.R158P has been determined as a pathogenic mutation in other tumors, and NOTCH2 p.G329S was a novel mutation. We identified two novel mutations that may have contributed to the pathogenesis of the secondary tumor's development. The roles of the mutations remain unclear.

Cigarette Smoke Underlies the Pathogenesis of Palmoplantar Pustulosis via an IL-17A-Induced Production of IL-36γ in Tonsillar Epithelial Cells.

Palmoplantar pustulosis (PPP) is characterized by sterile pustules on the palms and soles. A strong association between PPP and tobacco smoking has been reported, and it has been speculated that the IL-17A pathway may play an important role in PPP. Recent studies have suggested that IL-36 plays a pivotal role in the pathogenesis of psoriasis and its subtypes. The relationships among IL-36, smoking, and PPP have not been examined. Here, we investigated the relationships among the smoking index, severity of the clinical condition of PPP, and in vitro dynamics of IL-36 in human tonsillar epithelial cells under the condition of exposure to a cigarette smoke extract. The results demonstrated that the Palmoplantar Pustulosis Area and Severity Index was strongly and positively correlated with the smoking index in female patients. Immunohistochemical examinations showed that IL-36γ was highly expressed in tonsillar epithelial cells from patients with PPP but not in those from patients with recurrent tonsillitis without PPP. The in vitro study revealed that IL-17A synergistically induced a release of IL-36γ under cigarette smoke extract exposure. These results suggest that local production of IL-36γ by epithelial cells induced by cigarette smoke exposure plays an important role in the pathogenesis of PPP.

Elucidation of Melanogenesis Cascade for Identifying Pathophysiology and Therapeutic Approach of Pigmentary Disorders and Melanoma.

Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis. Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals. Fully melanized melanosomes are delivered to keratinocytes of the skin and hair. The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU). Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis. Diseases involving alterations of EMU show various forms of pigmentation phenotypes. This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade.

Regular sweating activities for the treatment of cholinergic urticaria with or without acquired idiopathic generalized anhidrosis.

Cholinergic urticaria (CholU) decreases affected individuals' quality of life because they must avoid stimuli including exercise and hot bathing. Although case reports have indicated that regular sweating activities are effective for CholU with hypohidrosis, little evidence is available. This retrospective medical record review examined CholU patients who received any form of treatment at our hospital. Twenty-seven cases (78% men; median age 22 years, range 12-70 years) were analyzed. Fourteen (52%) patients had acquired idiopathic generalized anhidrosis (AIGA). Among the 12 patients receiving sweating therapy (4 with, 8 without AIGA), improvement of symptoms was confirmed in 11 (92%; sweating therapy alone: n = 5, with H1 blocker: n = 5, with steroid pulse: n = 1) including 8 (67%) showing complete response (CR). In this sweating-therapy group, CR was achieved by six of the eight (75%) patients without AIGA and two of the four (50%) patients with AIGA. Among the 15 patients without sweating therapy, symptom improvement was observed in 9 (60%; steroid pulse: n = 7, H1 blocker: n = 2) including 1 (7%) achieving CR. Sweating therapy was safely undertaken except in one case in which the patient showed angioedema and anaphylaxis. Regular sweating activities could be a potential therapeutic option for CholU patients.

Association between PD-L1 expression and lymph node metastasis in cutaneous squamous cell carcinoma.

AIM: To clarify the relationship between programmed cell death ligand 1 (PD-L1) expression in cutaneous squamous cell carcinoma (cSCC) and clinicopathological variables. METHODS: We examined PD-L1 expression in tumor cells (TCs) and tumor infiltrating immune cells (ICs) in 46 cases of cSCC by immunohistochemistry. In each case, we employed two methods-intensity and proportion scores-to evaluate PD-L1 expression in TCs. For the evaluation of PD-L1 expression in ICs, only the proportion score was used. Association between PD-L1 expression and clinicopathological variables was analyzed using Fisher's exact test. RESULTS: High intensity scores in TCs were observed in 18 of the 46 cases (39.1%) and low intensity scores were observed in 28 cases (60.9%). Applying the proportions, using cut-off values of ≥1% and 50%, positive scores in TCs were observed in 36 (78.3%) and 20 cases (43.5%), respectively. PD-L1-positive ICs were observed in 29 (63%) and seven cases (15.2%), using cut-off values of ≥1% and 10%, respectively. The high intensity scores in TCs correlated with lymph node metastasis (P = 0.008) and female gender (P = 0.017), although positive proportions in TCs or ICs were not significantly related to lymph node metastasis. A multivariate analysis showed that high intensity of PD-L1 expression in TCs was an independent risk factor for lymph node metastasis. CONCLUSIONS: The results suggested that high intensity of PD-L1 expression in TCs is associated with lymph node metastasis in cSCC.