Genomic landscape of cutaneous, acral, mucosal, and uveal melanoma in Japan: analysis of clinical comprehensive genomic profiling data.

BACKGROUND: Cutaneous melanoma (CM) is the most common type in Caucasians, while acral melanoma (AM) and mucosal melanoma (MM), which are resistant to immunotherapies and BRAF/MEK-targeted therapies, are more common in East Asians. Genomic profiling is essential for treating melanomas, but such data are lacking in Japan. METHODS: Comprehensive genomic profiling data compiled in the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) were analyzed. RESULTS: A total of 380 melanomas was analyzed, including 136 CM, 46 AM, 168 MM, and 30 uveal melanoma (UM). MM included conjunctival, sinonasal, oral, esophageal, anorectal, and vulvovaginal melanomas. No significant difference in the median tumor mutational burden (TMB) of CM (3.39 mutations/megabase), AM (2.76), and MM (3.78) was the key finding. Microsatellite instability-high status was found in one case. BRAF V600E/K was found in only 45 patients (12%). Key driver mutations in CM were BRAF (38%), NRAS (21%), NF1 (8%), and KIT (10%), with frequent copy number alterations (CNAs) of CDKN2A, CDKN2B, and MYC. AM was characterized by altered KIT (30%), NRAS (26%), and NF1 (11%) and CDKN2A, CDKN2B, CDK4, MDM2, and CCND1 CNAs. MM was characterized by altered NRAS (24%), KIT (21%), and NF1 (17%) and MYC, KIT, and CDKN2A CNAs, with differences based on anatomical locations. UM bore GNAQ or GNA11 driver mutations (87%) and frequent mutations in SF3B1 or BAP1. CONCLUSION: The distinct genomic profiling in Japanese patients, including lower TMB, compared to Caucasians, is associated with poorer treatment outcomes. This result underscores the need for more effective therapeutic agents.

Mosaic SUFU mutation associated with a mild phenotype of multiple hereditary infundibulocystic basal cell carcinoma syndrome.

Multiple hereditary infundibulocystic basal cell carcinoma syndrome (MHIBCC), an autosomal dominant disorder caused by variants in SUFU, is characterized by numerous infundibulocystic basal cell carcinomas (IBCCs). In this report, we present a possible case of mosaic MHIBCC. A 57-year-old woman underwent the removal of four papules on her face, which were diagnosed as IBCCs. Exome sequencing revealed a SUFU c.1022+1G>A mutation within the skin tumor. The same mutation was detected in her blood but at a lower allele frequency. TA cloning revealed that the allele frequency of the mutation in the blood was 0.07. Additionally, tumor assessment revealed loss of heterozygosity (LOH) in chromosome 10, including the SUFU locus. These results indicate the patient had mosaicism for the SUFU mutation in normal tissues, aligning with the mosaic MHIBCC diagnosis. This, combined with LOH, likely contributed to IBCC development. Mosaic MHIBCC may present with milder symptoms. However, it may still increase the risk of developing brain tumors and more aggressive basal cell carcinoma. The possibility of mosaicism should be investigated in mild MHIBCC cases, where standard genetic tests fail to detect SUFU germline variants.

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 µM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.

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.

3D Spheroid Configurations Are Possible Indictors for Evaluating the Pathophysiology of Melanoma Cell Lines.

To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM.

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.

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.

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.

IL-13 modulates ∆Np63 levels causing altered expression of barrier- and inflammation-related molecules in human keratinocytes: A possible explanation for chronicity of atopic dermatitis.

BACKGROUND: Barrier disruption and an excessive immune response in keratinocytes are now considered to have important roles in the pathophysiology of atopic dermatitis (AD). Furthermore, disturbed keratinocyte differentiation is considered to underlie AD. ΔNp63, a p53-like transcription factor, is a major regulator of keratinocyte differentiation. However, the functional significance of ΔNp63 in AD has not been clarified. OBJECTIVE: In this study, we aimed to investigate the influence of the type 2 inflammatory environment on ΔNp63 expression and AD-associated molecules regulated by ΔNp63 in keratinocytes. METHODS: The immunohistochemical expression profiles of ΔNp63 and AD-related molecules were evaluated in human skin tissue. The function of ΔNp63 in the regulation of AD-related molecules and the influence of the type 2 inflammatory environment on ΔNp63 expression were investigated using human primary keratinocytes. Expression of ΔNp63 was manipulated using the RNA interfering method. RESULTS: In healthy skin tissue, we observed an inverse expression pattern between ∆Np63 and some barrier-related proteins including filaggrin, caspase-14, claudin-1, and claudin-4. ΔNp63 regulated expression of these genes and proteins. In addition, production of IL-1ß and IL-33, pro-inflammatory cytokines, was modulated by ΔNp63. Furthermore, prolonged IL-13 exposure increased the thickness of the three-dimensional culture of keratinocytes. IL-13 interfered with ΔNp63 downregulation during calcium-induced keratinocyte differentiation. IL-13 modulated some barrier-related and inflammation-related molecules, which were regulated by ΔNp63. CONCLUSIONS: We have shown that ΔNp63 modulated AD-related barrier and inflammatory molecules. In addition, ΔNp63 expression was affected by IL-4/IL-13. IL-13-ΔNp63 axis would integrate two major factors of AD pathogenesis: dysregulated barrier and inflammation.

Usefulness of neuron-specific enolase as a serum marker of metastatic melanoma.

Treatment strategies for advanced melanoma are dramatically changing, due to immune-checkpoint inhibitors and BRAF/MEK inhibitors. Nevertheless, reliable serum markers for evaluation of treatment responses and the outcome are still limited. Some previous reports suggested that serum neuron-specific enolase (sNSE) may be a useful marker for melanoma; however, its usefulness is controversial. Moreover, NSE has not been examined in vitro by using melanoma cell lines. We retrospectively evaluated sNSE and serum lactate dehydrogenase (sLDH) levels at the initial diagnosis and during therapy in 33 melanoma patients of various stages. We analyzed the NSE concentrations in cell lysates and supernatants from melanoma cell lines by enzyme-linked immunosorbent assay. The median sNSE was significantly higher in stage IV patients compared with stages I/II and III (16.3, 12.7 and 12.1 ng/mL, respectively). sNSE was elevated in 20% (2/10) of stage III and 61.1% (11/18) of stage IV patients but not in stages I/II. sNSE and sLDH tended to correspond to the total tumor volume (P = 0.48 and 0.58; 95% confidence intervals, 0.005-0172 and 0.776-0.836, respectively). The coincidence rate of sNSE and sLDH in stage IV at the initial diagnosis was 11 of 18 (61.1%). Of the remaining patients, elevated sNSE but not sLDH was observed in five patients (27.8%) and elevated sLDH but not sNSE was observed in two (11.1%). Four of the five patients showing elevated sNSE and normal sLDH were of the mucosal type. NSE was detected in both supernatant and cell lysate of all four melanoma cell lines (0.30-237.32 ng/mL and 137-483.04 ng/mg, respectively). Two cell lines with a high supernatant NSE level contained many dead cells in the supernatant. The combination of sNSE and sLDH could contribute to the early detection of distant metastasis and disease condition evaluations for advanced melanoma patients.

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.