GWAS for systemic sclerosis identifies six novel susceptibility loci including one in the Fcγ receptor region.

Here we report the largest Asian genome-wide association study (GWAS) for systemic sclerosis performed to date, based on data from Japanese subjects and comprising of 1428 cases and 112,599 controls. The lead SNP is in the FCGR/FCRL region, which shows a penetrating association in the Asian population, while a complete linkage disequilibrium SNP, rs10917688, is found in a cis-regulatory element for IRF8. IRF8 is also a significant locus in European GWAS for systemic sclerosis, but rs10917688 only shows an association in the presence of the risk allele of IRF8 in the Japanese population. Further analysis shows that rs10917688 is marked with H3K4me1 in primary B cells. A meta-analysis with a European GWAS detects 30 additional significant loci. Polygenic risk scores constructed with the effect sizes of the meta-analysis suggest the potential portability of genetic associations beyond populations. Prioritizing the top 5% of SNPs of IRF8 binding sites in B cells improves the fitting of the polygenic risk scores, underscoring the roles of B cells and IRF8 in the development of systemic sclerosis. The results also suggest that systemic sclerosis shares a common genetic architecture across populations.

Genomic landscape of circulating tumour DNA in metastatic extramammary Paget's disease.

Although cancer personalized profiling by deep sequencing (CAPP-Seq) of cell-free DNA (cfDNA) has gained attention, the clinical utility of circulating tumour DNA (ctDNA) in extramammary Paget's disease (EMPD) has not been investigated. In this study, genomic alterations in the cfDNA and tumour tissue DNA were investigated in seven patients with metastatic EMPD. CAPP-Seq revealed mutations in 18 genes, 11 of which have not yet been reported in EMPD. The variant allele frequency of some of the mutated genes reflected the disease course in patients with EMPD. In one patient, the mutation was detected even though imaging findings revealed no metastasis. In another patient with triple EMPD (genital area and both axilla), cfDNA sequencing detected the mutation in a rib metastatic lesion, which was also detected in both axilla lesions but not the genital region. Investigations of the ctDNA may be useful towards the elucidation of clonal evolution in EMPD.

A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells.

Turning non-inflamed (cold) tumors into inflamed (hot) tumors is important for maximizing the effect of immune checkpoint inhibitors (ICIs) against malignancies. We showed that lactate, a product of the Warburg effect, inhibited the efficacy of ICIs and suppressed IL-12 p40 expression in dendritic cells (DCs) through reducing NF-κB p65, p50, and c-Rel DNA-binding activity to the IL-12 p40 promoter. Additionally, lactate promoted the expression of early growth response protein 1 (EGR1), whose expression was increased in human invasive melanoma compared with non-invasive melanoma. We also found that EGR1 interacts with serum response factor (SRF) and represses the expression of CD80 in DCs. These findings suggest that lactate and its induced EGR1 are key factors that turn hot tumors into cold tumors and may represent targets in cancer treatment with ICIs.

Liquid biopsy-based analysis by ddPCR and CAPP-Seq in melanoma patients.

BACKGROUND: The development of BRAF/MEK inhibitors in patients with metastatic melanoma harboring BRAF mutations has garnered attention for liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) or next-generation sequencing methods. OBJECTIVE: To investigate gene mutations in tumor DNA and cfDNA collected from 43 melanoma patients and evaluate their potential as biomarkers. METHODS: ddPCR and CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) techniques were performed to detect gene mutations in plasma cfDNA obtained from patients with metastatic melanoma. RESULTS: Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in the plasma cfDNA, and the results were partially consistent with the results of those identified in the tissues. Among the variants examined, copy numbers of MET mutations were consistent with the disease status in two melanoma patients. CONCLUSION: Liquid biopsy using CAPP-Seq and ddPCR has the potential to detect tumor presence and mutations, especially when tissue biopsies are unavailable. MET mutations in cfDNA may be a potential biomarker in patients with metastatic melanoma.

Overexpression of Janus kinase 2 protein in extramammary Paget's disease.

Extramammary Paget's disease is a rare malignant tumor of the skin that occurs primarily in the genitocrural region. Although the prognosis of extramammary Paget's disease with distant metastasis is poor, an effective therapy has not been established. Because Janus kinase 2 has attracted attention as a therapeutic target in several cancers, we investigated the expression of the Janus kinase 2 protein and the relationship between its level of expression and clinical significance in 53 patients with extramammary Paget's disease in our hospital. Immunohistochemistry showed that most extramammary Paget's disease tissues were positive for Janus kinase 2 (50/53, 94.3%), and the immunostaining intensity of Janus kinase 2 was correlated with the degree of invasiveness, lymph node metastasis and distant metastasis. Based on these findings, Janus kinase 2 may be a promising therapeutic target in extramammary Paget's disease.

Immunotherapy with 4-1BBL-Expressing iPS Cell-Derived Myeloid Lines Amplifies Antigen-Specific T Cell Infiltration in Advanced Melanoma.

We have established an immune cell therapy with immortalized induced pluripotent stem-cell-derived myeloid lines (iPS-ML). The benefits of using iPS-ML are the infinite proliferative capacity and ease of genetic modification. In this study, we introduced 4-1BBL gene to iPS-ML (iPS-ML-41BBL). The analysis of the cell-surface molecules showed that the expression of CD86 was upregulated in iPS-ML-41BBL more than that in control iPS-ML. Cytokine array analysis was performed using supernatants of the spleen cells that were cocultured with iPS-ML or iPS-ML-41BBL. Multiple cytokines that are beneficial to cancer immunotherapy were upregulated. Peritoneal injections of iPS-ML-41BBL inhibited tumor growth of peritoneally disseminated mouse melanoma and prolonged survival of mice compared to that of iPS-ML. Furthermore, the numbers of antigen-specific CD8+ T cells were significantly increased in the spleen and tumor tissues treated with epitope peptide-pulsed iPS-ML-41BBL compared to those treated with control iPS-ML. The number of CXCR6-positive T cells were increased in the tumor tissues after treatment with iPS-ML-41BBL compared to that with control iPS-ML. These results suggest that iPS-ML-41BBL could activate antigen-specific T cells and promote their infiltration into the tumor tissues. Thus, iPS-ML-41BBL may be a candidate for future immune cell therapy aiming to change immunological "cold tumor" to "hot tumor".

Nucleosome assembly protein 1-like 4, a new therapeutic target for proliferation and invasion of melanoma cells.

BACKGROUND: Melanoma is one of the deadliest skin cancers. The treatment of advanced melanoma has been dramatically improved by immune checkpoint inhibitors and targeted therapies. However, many patients still do not respond to these therapies. OBJECTIVE: To investigate whether NAP1L4 can be a new therapeutic target for melanoma. METHODS: Immunohistochemical analysis of human nevus and melanoma tissues was performed. Real-time RT-PCR and immunoblotting were performed using human samples and melanoma cell lines. Next, we examined the effect of NAP1L4 knockdown in melanoma cell lines using cell migration and invasion assays. To investigate the molecular mechanism related to these results, immunoblotting of p21 and Slug was examined. MMP-2 and MMP-9 activity assays were also performed. Further, pathway analysis between NAP1L4 and MMP-2 was performed. Finally, the effects of NAP1L4 knockdown on cell proliferation, apoptosis, and cell cycle were analyzed. RESULTS: NAP1L4 was overexpressed in melanoma tissues compared to the nevus tissue. NAP1L4 knockdown reduced melanoma cell migration and invasion. NAP1L4 knockdown upregulated p21 and downregulated Slug expression in melanoma cells. NAP1L4 knockdown decreased the active levels of MMP-2 in the supernatant from melanoma cells. NAP1L4 knockdown inhibited apoptosis in camptothecin-induced DNA damage, induced cell cycle arrest at the G1/S phase, and inhibited cell proliferation. CONCLUSIONS: NAP1L4 may play a role in cell migration and invasion in melanoma cells through the regulation of Slug. We propose that NAP1L4 can be a new therapeutic target for proliferation and invasion of melanoma cells.

Existence of Staphylococcus aureus correlates with the progression of extramammary Paget's disease: potential involvement of interleukin-17 and M2-like macrophage polarization.

BACKGROUND: The microbiome plays an important role in the tumour microenvironment (TME). OBJECTIVES: In this study, we investigated the clinical significance of the microbiota in extramammary Paget's disease (EMPD). MATERIALS & METHODS: Patients with EMPD, treated between March 2007 and September 2019 at Kumamoto University Hospital, were investigated retrospectively. Inclusion criteria included: histological diagnosis of EMPD, inspection of the bacterial culture of the cancer lesion using swab sampling, and availability of sufficient tissue in paraffin blocks for immunohistochemistry. For the latter, primary antibodies against IL-17, CD163 and ionized calcium-binding adapter molecule 1 (Iba1) were used. RESULTS: Bacterial cultures of the cancer lesion revealed that Staphylococcus aureus (S. aureus) was highly prevalent in EMPD patients, with dermal invasion or lymph node metastasis, compared to patients without these findings. Furthermore, the number of IL-17-positive cells and CD163-positive M2-like macrophages (pro-tumour macrophages) were increased in EMPD tissues with S. aureus. Moreover, the number of IL-17-producing cells in EMPD tissues positively correlated with the accumulation of CD163-positive M2-like macrophages. In addition, the percentage of CD163-positive cells within Iba-1-positive macrophages (total macrophages) was also significantly elevated in EMPD tissues with S. aureus. CONCLUSION: Based on these findings, S. aureus may exacerbate the pathological condition of EMPD via the accumulation of IL-17 and M2-like macrophages.

Effect of adalimumab on axial manifestations in Japanese patients with psoriatic arthritis: a 24 week prospective, observational study.

OBJECTIVES: PsA is characterized by enthesitis, synovitis and osseous involvement in the peripheral and axial joints. Few studies have examined axial involvement in PsA using imaging techniques. Here we examined axial involvement in PsA patients using MRI. In addition, we determined the efficacy of 24 week adalimumab treatment in improving the MRI findings of spondylitis and sacroiliitis. METHODS: This was a prospective, open-label, single-arm study in patients with PsA. Adalimumab was administered to patients for a total of 24 weeks. MRI examinations were conducted at baseline and at week 24 of adalimumab treatment. RESULTS: Thirty-seven patients with PsA were included in this study. Spondylitis was observed in at least one site of the positive scan in 91% (n = 31) of patients with PsA. The number of arthritic sites in the cervical, thoracic and lumbar regions of the spine was 48, 67 and 53, respectively. All patients had MRI-determined sacroiliitis of grade ≥1 severity while 28 patients (82%) had grade ≥2 sacroiliitis in at least one sacroiliac region. Sacroiliac arthritis was statistically more severe on the right side than on the left side (P < 0.05). In 34 patients with PsA, the thoracic spine was the most common site of spondylitis. In addition, 24 week adalimumab treatment led to an improvement in the mean number of spondylitis sites and the mean grade of sacroiliitis. CONCLUSION: Treatment with adalimumab for 24 weeks resulted in improvement in spondylitis and sacroiliitis.

Initial predictors of skin thickness progression in patients with diffuse cutaneous systemic sclerosis: Results from a multicentre prospective cohort in Japan.

OBJECTIVE: To identify initial parameters that predict worsening of skin thickening in patients with diffuse cutaneous systemic sclerosis (dcSSc) using a multicentre, prospective, observational cohort in Japan. METHODS: A total of 171 patients with dcSSc were selected from a prospective cohort database based on the following criteria: dcSSc, modified Rodnan total skin thickness score (mRSS) ≥7, disease duration <60 months, and valid mRSS data at one year. Worsening of skin thickness was defined as an increase in mRSS ≥3 points and an increase ≥25% from baseline to one year. Initial demographic and clinical parameters useful for predicting the progression of skin thickness were identified using univariate and multivariable analysis, and prediction models of skin thickening progression were built based on combinations of independent predictive parameters. RESULTS: Only 23 patients (13.5%) experienced worsening mRSSs at one year. Short disease duration, low mRSS, absence of nailfold bleeding, arthritis, and a high erythrocyte sedimentation rate at diagnosis were identified as predictors of subsequent worsening of the mRSS even after adjusting for the treatment. Assessment of the best predictive model revealed that patients with a disease duration ≤12 months and mRSS ≤19 had a risk of mRSS worsening within one year, with a sensitivity of 73.9% and specificity of 81.1%. CONCLUSION: Identification of predictors of subsequent worsening of skin thickness in dcSSc patients is useful for identifying patients who require intensive treatment with potential disease-modifying agents and for improving clinical trial design by characterizing eligible progressors in the Japanese population.

Fosravuconazole to treat severe onychomycosis in the elderly.

Fosravuconazole is a novel oral antifungal drug developed in Japan and used to treat tinea unguium since 2018. Its excellent oral absorbability and systemic bioavailability has enabled short-duration therapy of 3 months. Furthermore, no concomitant drugs are contraindicated due to the presence of the mild inhibitor of cytochrome P450 enzyme which is responsible for polypharmacy adverse effects. Therefore, it can be safely administrated to elderly patients. Elderly patients (≥65 years old) with severe onychomycosis (≥50% nail involvement) were treated with oral fosravuconazole 100 mg once daily for 12 weeks. The rate of involvement improved from 86.6% to 28.1% (P < 0.01). The efficacy (i.e. percentage of those rated as "improved" and better) and cure rate was 83.8% (31/37) and 29.7% (11/37), respectively. Furthermore, when focusing on the thin nail group (<3 mm), the efficacy and cure rate was 88.2% (15/17) and 58.8% (10/17), respectively. Although the serum γ-glutamyltransferase levels increased in 21.6% (8/37), all patients recovered without any specific treatments.

Clinical course of Japanese patients with early systemic sclerosis: A multicenter, prospective, observational study.

OBJECTIVES: To investigate the clinical course of Japanese patients with early diffuse cutaneous systemic sclerosis (dcSSc) and early SSc with interstitial lung disease (ILD). METHODS: We prospectively analyzed the clinical features of 207 Japanese patients with early dcSSc (n = 150) and limited cutaneous SSc (lcSSc) with ILD (n = 57) in 10 medical centers every year for 7 consecutive years. RESULTS: Mean modified Rodnan total skin thickness score (mRSS) was 18.3 and 67.4% of the cohort had ILD. Most patients started immunosuppressive therapy and vasodilators during 7 years (83.4% and 87.9%, respectively). Mean value of mRSS of total patients was significantly reduced from the initial registration after the first year. However, other parameters for physical function associated with skin sclerosis including fist closure, hand extension, and oral aperture were not so ameliorated during the study period. Health Assessment Questionnaire-disability index and serum KL-6 levels were constant throughout the course. Percent vital capacity and the presence of ILD, clinically suspected pulmonary arterial hypertension, and digital ulcers were gradually exacerbated during the period. CONCLUSION: In Japanese early dcSSc patients and SSc patients with ILD, mRSS was continuously reduced during 7 years of follow-up, but there was little improvement of physical disability and organ involvement.

Ungual hyalohyphomycosis caused by Fusarium cugenangense.

Fusarium onychomycosis is uncommon in the temperate climate zone of Japan. Based on the morphological characteristics and a gene analysis, we diagnosed a patient with ungual hyalohyphomycosis caused by Fusarium cugenangense belonging to the F oxysporum complex. This intractable disease was cured by 6-month treatment with efinaconazole 10% solution.

miR-524-5p reduces the progression of the BRAF inhibitor-resistant melanoma.

BRAF inhibitors were approved for the treatment of BRAF-mutant melanoma. However, most patients acquire the resistance to BRAF inhibitors after several months of treatment. miR-524-5p is considered as a tumor suppressor in many cancers, including melanoma. In this study, we investigated the biological functions of miR-524-5p in melanoma with acquired resistance to BRAF inhibitor and evaluated the endogenous miR-524-5p expression as a biomarker for melanoma. The results showed that the expression of miR-524-5p was 0.481-fold lower in melanoma tissues (n = 117) than in nevus tissues (n = 40). Overexpression of miR-524-5p significantly reduced proliferative, anchorage-independent growth, migratory and invasive abilities of BRAF inhibitor-resistant melanoma cells. Moreover, the introduction of miR-524-5p led to a reduced development of BRAF inhibitor-resistant melanoma in vivo. Remarkably, the MAPK/ERK signaling pathway was decreased after treatment with miR-524-5p. Furthermore, next-generation sequencing analysis implied that the complement system, leukocyte extravasation, liver X receptor/retinoid-X-receptor activation, and cAMP-mediated signaling may be related to miR-524-5p-induced pathways in the resistant cells. The miR-524-5p level was higher on average in complete response and long-term partial response patients than in progressive disease and short-term partial response patients treated with BRAF inhibitors. Our results proposed that miR-524-5p could be considered as a target for treatment BRAF inhibitor-resistant melanoma and a prognostic marker in the response of patients to BRAF inhibitors for melanoma.