Azusa Miyashita

Myasthenic crisis and polymyositis induced by one dose of nivolumab

An 80‐year‐old man, who developed multiple lymph node and skin metastasis of malignant melanoma, received nivolumab monotherapy. Two weeks after the first dose, he experienced anorexia and fatigue, and suffered from progressive, severe dyspnea and muscle weakness. We diagnosed him with myocarditis, myositis, and myasthenic crisis induced by nivolumab. We commenced steroid therapy, immune absorption therapy, plasma exchange therapy, and i.v. immunoglobulin therapy, and succeeded in saving his life. Because his serum level of anti‐acetylcholine receptor antibodies in a sample collected before nivolumab treatment were positive and were elevated significantly after nivolumab, we suspected that nivolumab triggered a severe autoimmune response, which progressed subclinical myasthenia gravis to myasthenic crisis. We carried out T cell receptor repertoire analysis using next‐generation sequencing technologies and identified infiltration of clonally expanded T cell populations in the skeletal muscle after nivolumab treatment, implying a very strong T cell immune response against muscular cells. To avoid severe immune‐related adverse events, the exclusion of patients with subclinical autoimmune disease is very important for treatment with immune checkpoint inhibitors.

Sensitive detection of melanoma metastasis using circulating microRNA expression profiles.

Numerous studies have indicated that the serum levels of microRNAs are useful for the diagnosis or evaluation of activity in human diseases. However, determining the level of only one of the nearly 2000 microRNAs identified so far may be less significant. Accordingly, we examined the possibility that the expression pattern of multiple microRNAs in each patient may be a more reliable disease marker for melanoma, especially metastatic disease, focusing on the interaction among microRNAs. Six microRNAs (miR-9, miR-145, miR-150, miR-155, miR-203, and miR-205) were evaluated using real-time PCR in 11 patients with metastatic melanoma and in 16 patients without melanoma. The expression of the six microRNAs was significantly different between the patients with metastasis and those without it. MiR-9 and miR-205 and miR-203 and miR-205 showed significant correlations, and the combination of miR-9, miR-145, miR-150, miR-155, and miR-205 was more sensitive than when each miR was used individually to distinguish the patients with metastasis from those without it. This is the first report demonstrating the expression profiles of multiple microRNAs in melanoma patients. Clarifying the involvement of the microRNA network in the pathogenesis of melanoma may contribute to the development of new diagnostic tools and to advancing the understanding of this disease.

Intratumoral expression levels of PD-L1, GZMA, and HLA-A along with oligoclonal T cell expansion associate with response to nivolumab in metastatic melanoma.

ABSTRACT Immune checkpoint inhibitors blocking the interaction between programmed death-1 (PD-1) and PD-1 ligand-1 (PD-L1) are revolutionizing the cancer immunotherapies with durable clinical responses. Although high expression of PD-L1 in tumor tissues has been implicated to correlate with the better response to the anti-PD-1 therapies, this association has been controversial. In this study, to characterize immune microenvironment in tumors, we examined mRNA levels of immune-related genes and characterized T cell repertoire in the tumors of 13 melanoma patients before and after nivolumab treatment. We found that, in addition to the PD-L1 (p = 0.03), expression levels of PD-1 ligand-2 (PD-L2), granzyme A (GZMA) and human leukocyte antigen-A (HLA-A) in the pre-treatment tumors were significantly higher (p = 0.04, p = 0.01 and p = 0.006, respectively) in responders (n = 5) than in non-responders (n = 8). With nivolumab treatment, tumors in responders exhibited a substantial increase of CD8, GZMA and perforin 1 (PRF1) expression levels as well as increased ratio of TBX21/GATA3, suggesting dominancy of helper T cell type 1 (Th1) response to type 2 (Th2) response. T cell receptor β (TCR-β) repertoire analysis revealed oligoclonal expansion of tumor-infiltrating T lymphocytes (TILs) in the tumor tissues of the responders. Our findings suggest that melanoma harboring high PD-1 ligands (PD-L1 and PD-L2), GZMA and HLA-A expression may respond preferentially to nivolumab treatment, which can enhance Th1-skewed cellular immunity with oligoclonal expansion of TILs.

The rs2910164 G>C polymorphism in microRNA-146a is associated with the incidence of malignant melanoma.

MicroRNA-146a (miR-146a) is one of the microRNAs (miRNAs) implicated in the pathogenesis of various cancers. Recently, single nucleotide polymorphisms (SNPs) located in miRNAs themselves, so-called MIRSNPs, have attracted attention because of their possible involvement in the pathogenesis of various diseases. Such MIRSNPs may play functional roles because of the alteration of the miRNA. In this study, we investigated whether MIRSNP rs2910164 in miR-146a is involved in the pathogenesis of malignant melanoma (MM). DNA samples were collected from 50 patients with MM and 107 controls and genotyped by a PCR-restriction fragment. In patients with MM, the genotype distributions were 15 CC (30.0%), 35 CG (70.0%), and 0 GG (0.0%). The CG genotype was significantly increased in the patients compared with the controls (P=0.02). The minimum free energy between miR-146a and its complementary strand with the G allele was determined to be −26.8 kcal/mol, whereas that of the C allele was −24.0 kcal/mol, indicating that the change from C to G may increase the stability of the miR-146a. However, there was no significant difference between the CC and the CG genotypes in terms of the relative expression levels of miR-146a. Human melanoma cell lines with the G allele showed significantly higher proliferation, migration, and invasion than those with the C allele. In conclusion, miR-146a may be involved in the pathogenesis of MM, and individuals with the CG genotype have an increased risk of developing MM.

Prognostic Significance of CD169+ Lymph Node Sinus Macrophages in Patients with Malignant Melanoma

Prognostic indicators are needed for malignant melanoma. The presence of high densities of CD169+ macrophages in the draining lymph nodes of patients significantly correlates with CTL infiltration and longer overall survival, providing a potentially useful biomarker. CD169 (sialoadhesin) is a sialic acid receptor that is specifically expressed on macrophages, including lymph node sinus macrophages. Animal studies suggest that CD169+ macrophages in lymph nodes have properties in preventing cancers. In order to determine the significance of CD169+ macrophages in patients with malignant melanoma, we evaluated tissue samples from 93 patients to investigate CD169 expression in regional lymph nodes (RLN) and determine the relationship of this expression with overall survival and various clinicopathologic factors. Higher densities of CD169+ cells were significantly associated with longer overall survival (P = 0.001). A multivariate analysis showed that the density of CD169+ cells was an independent prognostic factor, with higher densities correlating with higher density of CD8+ cytotoxic T cells within tumor sites. High CD169 expression in macrophages could be stimulated by IFNα in vitro, and in RLNs, IFNα-producing macrophages and CD303+ plasmacytoid dendritic cells were identified surrounding CD169+ macrophages. These data suggest that IFNα-stimulated CD169+ macrophages in RLNs are closely involved in T-cell–mediated antitumor immunity and may be a useful marker for assessing the clinical prognosis and monitoring antitumor immunity in patients with malignant melanoma. Cancer Immunol Res; 3(12); 1356–63. ©2015 AACR.

Evaluation of sentinel node biopsy for cutaneous squamous cell carcinoma.

Sentinel lymph node biopsy (SLNB) is a standard care for cutaneous melanoma but its role in cutaneous squamous cell carcinoma (SCC) has not been established. Clinical data was obtained from 54 patients with SCC who received SLNB with the usage of blue dye and radioisotope colloid methods. The positive rate of SLNB in SCC was 7.4%. If the cases were limited to more than T2, the positive rate was 12.9%. Three of 41 patients who was estimated negative LN metastasis by the preoperative tests had micrometastasis (7.3%). Among 13 patients who were suggested to have metastasis in the preoperative tests, only one patient had histological metastasis. One patient with SCC located in the lower lip showed negative SLNB and subsequently developed node recurrence. In conclusion, the efficacy of SLNB in SCC is comparable to that of melanoma in the positive rate. There are two kinds of benefit, avoidance of unnecessary complete lymph node dissection and early detection of metastasis.

Investigation of FOXM1 as a Potential New Target for Melanoma.

Recent studies have shown that immunotherapies and molecular targeted therapies are effective for advanced melanoma. Non-antigen-specific immunotherapies such as immunocheckpoint blockades have been shown to be effective in the treatment of advanced melanoma. However, the response rates remain low. To improve their efficacy, they should be combined with antigen-specific immunotherapy. Elevated expression of the transcription factor, Forkhead box M1 (FOXM1), has been reported in various human cancers, and it has been shown to have potential as a target for immunotherapy. The purpose of this study was to investigate the FOXM1 expression in human melanoma samples and cell lines, to evaluate the relationship between the FOXM1 expression and the clinical features of melanoma patients and to investigate the association between the FOXM1 and MAPK and PI3K/AKT pathways in melanoma cell lines. We conducted the quantitative reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines, and investigated melanoma and nevus tissue samples by qRT-PCR and immunohistochemistry. We performed MEK siRNA and PI3K/AKT inhibitor studies and FOXM1 siRNA studies in melanoma cell lines. We found that FOXM1 was expressed in all of the melanoma cell lines, and was expressed in 49% of primary melanomas, 67% of metastatic melanomas and 10% of nevi by performing immunohistochemical staining. Metastatic melanoma samples exhibited significantly higher mRNA levels of FOXM1 (p = 0.004). Primary melanomas thicker than 2 mm were also more likely to express FOXM1. Patients whose primary melanoma expressed FOXM1 had a significantly poorer overall survival compared to patients without FOXM1 expression (p = 0.024). Downregulation of FOXM1 by siRNA significantly inhibited the proliferation of melanoma cells, and blockade of the MAPK and PI3K/AKT pathways decreased the FOXM1 expression in melanoma cell lines. In conclusion, FOXM1 is considered to be a new therapeutic target for melanoma.

Serum levels of leptin receptor in patients with malignant melanoma as a new tumor marker

Leptin is known to be abnormally expressed in a variety of cancers, and leptin receptors have been reported to be expressed on human melanoma cells. In this study, we evaluated the possibility that the serum levels of leptin receptor could be a tumor marker of malignant melanoma (MM). Serum samples were obtained from 71 patients with MM, and the serum levels of leptin receptor were measured by double‐determinant ELISA. Interestingly, serum levels of leptin receptor decreased gradually with the stages of MM, being highest at in situ and lowest at stage IV. There was also a trend of reverse correlation between tumor thickness and serum levels of leptin receptor. To our knowledge, this is the first report investigating the serum levels of leptin receptor in MM, and serum leptin receptor levels may be used as a useful tumor marker of MM.

The proportion of lymphocytic inflammation with CD123-positive cells in lupus erythematous profundus predict a clinical response to treatment.

Lupus erythematosus profundus is a rare inflammatory disorder of subcutaneous fat in patients with lupus ery-thematosus. Previous reports suggested that plasmacytoid dendritic cells, which expressed CD123 and CD303 antigens, play a central proinflammatory role in the patho-genesis of lupus erythematosus. To find the factors that determine the response to treatment, we analysed 23 skin specimens from the patients with lupus erythematosus profundus. The patients with considerable lymphocytic inflammation with high percentages of CD123+ cells in dermis and subcutaneous fat significantly responded to the systemic corticosteroid therapies. On the other hand, the patients with minor lymphocytic inflammation with low percentages of CD123+ cells showed poor response to treatments. The mean percentage of CD123+ cells in patients who showed good response to therapy was significantly higher than those that showed poor response (p = 0.027). These results suggest that the clinical response to treatment of lupus erythematosus profundus could be predicted from the histological features.

Case report of cutaneous protothecosis caused by Prototheca wickerhamii designated as genotype 2 and current status of human protothecosis in Japan

An 85‐year‐old Japanese woman presented with infiltrative erythema and ulceration on the extensor surface of her right forearm. Direct microscopic examination demonstrated spherical and morula‐like sporangia, while histopathology revealed numerous microorganisms with a mulberry‐like appearance in the dermis. Staining of the microorganisms also showed mulberry‐like sporangia that resembled the spokes of a wheel. The isolated yeast‐like microorganism had been identified as Prototheca wickerhamii genotype 2 in another independent study on the basis of its morphological, biochemical and genetic analysis. This case of protothecosis was recorded in Kyushu, Japan, and oral treatment with itraconazole 200 mg/day for 2 months was effective. Herein, we also summarize and analyze 39 cases of human protothecosis reported in Japan since the first record in 1983.