Eribulin for patients with metastatic extramammary Paget disease: Study protocol for a single-arm phase II trial.

Extramammary Paget disease (EMPD) is a rare cutaneous malignancy that predominantly affects the anogenital areas of the elderly. Although the efficacy of docetaxel and other cytotoxic agents for advanced EMPD has been reported in small retrospective case studies, no treatment has been proven effective in prospective clinical trials. We established the world's first in vivo EMPD experimental model (a patient-derived xenograft model). In our treatment experiment, xenograft tumours showed a remarkable response to eribulin. This study evaluates the efficacy of eribulin for patients with advanced EMPD. In October 2022, we started a single-arm phase II trial to evaluate the efficacy of eribulin as a treatment for adult patients with unresectable EMPD with measurable lesions. Enrolment in this clinical trial is open to patients with any prior treatment for EMPD. The primary endpoint is overall response rate; the secondary endpoints include disease control rate, overall survival, progression-free survival and adverse events. The study protocol was approved by the Ethics Committee of Hokkaido University and the other collaborating institutions. If the primary endpoint is met, it is our hope that eribulin will be regarded as a standard medication for patients with advanced EMPD.

Multiple courses of steroid pulse therapy are required in treating acquired idiopathic generalized anhidrosis patients with a large anhidrotic area: A retrospective study of 28 cases.

Patients with acquired idiopathic generalized anhidrosis (AIGA) demonstrate a sudden loss of sweating function without neurological or endocrine abnormalities. The main treatment is steroid pulse therapy. However, the number of courses required for improvement has been unclear. This study aims to clarify the factors associated with AIGA disease severity and with AIGA patients' responses to steroid pulse therapy. We retrospectively analysed the clinical information of 28 patients with AIGA in our department from the last 10 years. Univariate analysis revealed that patients with a large anhidrotic area need multiple courses of steroid pulse therapy.

Beneficial effects of nintedanib on cardiomyopathy in patients with systemic sclerosis: a pilot study.

OBJECTIVES: Nintedanib is an inhibitor of tyrosine kinases that has been shown to slow the progression of interstitial lung disease (ILD), including ILD associated with SSc. The aim of this study was to explore the effect of nintedanib on cardiomyopathy associated with systemic sclerosis (SSc). METHODS: Twenty consecutively hospitalized patients with SSc-ILD were enrolled and prospectively followed. The rate of change at 6 months in cardiac magnetic resonance (CMR) parametric mapping, including myocardial extracellular volume, was primarily evaluated. Other endpoints included changes in CMR functional parameters, echocardiographic parameters, modified Rodnan skin score, serum biomarkers and pulmonary function test. RESULTS: Nintedanib was administered in 10 patients, whereas the other 10 were treated without nintedanib or watched, according to ILD severity and progression. Baseline values of CMR parametric mapping were not different between the two groups. The rate of change at 6 months in myocardial extracellular volume was highly different, almost divergent, between the nintedanib group and the control group (-1.62% vs +2.00%, P = 0.0001). Among other endpoints, the change in right ventricular ejection fraction was significantly different between the two groups (P = 0.02), with a preferential change in the nintedanib group. CONCLUSION: Our data indicate beneficial signals of nintedanib on cardiomyopathy associated with SSc. The anti-fibrotic effect of nintedanib might not be limited to the lung.

Hair follicle stem cell progeny heal blisters while pausing skin development.

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

Prognostic Analysis of Patients With Extramammary Paget Disease Treated With Conservative Excision.

BACKGROUND: Extramammary Paget disease (EMPD) is a malignant skin tumor with a relatively good prognosis. The standard treatment is wide local resection or Mohs micrographic surgery. However, conservative excision may be a better option when radical wide local excision is difficult to perform due to the patients' mental or physical condition. There have been no studies on the prognosis of patients with EMPD who underwent conservative excision. OBJECTIVE: To compare the prognosis of conservative excision cases to wide excision cases of EMPD. MATERIALS AND METHODS: The authors retrospectively analyzed the clinical data of 69 cases of EMPD without metastases to lymph nodes or organs (11 cases treated with conservative excision, 58 cases treated with wide local excision) who underwent resection of the primary tumor from 2002 to 2022 in the Department of Dermatology at Hokkaido University Hospital. RESULTS: The log-rank test showed no significant differences in overall survival or metastasis-free survival between the wide excision group and the conservative excision group, although conservative surgery was often chosen in elderly patients or patients with lower performance status. CONCLUSION: This study suggests that conservative surgery should be considered as a treatment option for EMPD.

Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing.

Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM.

Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.

An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

Cyclin-dependent kinase 4/6 inhibitors suppress tumor growth in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare adnexal neoplasm commonly seen in the genital areas among the senior population. The prognosis of advanced EMPD is not favorable; thus, the development of potential treatments has long been sought. Cyclin-dependent kinase (CDK) 4/6 inhibitors such as abemaciclib and palbociclib have been proven effective against metastatic breast cancer; however, no studies have addressed CDK4/6 inhibitors as an EMPD treatment. We herein examine the efficacy of CDK4/6 inhibitors against an EMPD patient-derived xenograft (PDX) model. Abemaciclib (50 mg/kg/day) or palbociclib (120 mg/kg/day) was given orally to tumor-bearing NOD/Scid mice over a 3-week period. We also investigated the protein expression levels of CDK4/6 and cyclin D1 through immunohistochemical staining using EMPD clinical samples. Treatment with abemaciclib or palbociclib as a single agent was found to significantly suppress tumor growth in EMPD-PDX. The Ki-67-positive ratio of the treated EMPD-PDX tumors was significantly lower than that of the nontreated tumors. Clinically, the expression levels of CDK4 and cyclin D1 were significantly higher in the EMPD tumor cells than in the normal epidermis. Our results suggest that CDK4/6 inhibitors could be novel and potent therapeutics for the treatment of EMPD.

Adjuvant nivolumab therapy may not improve disease-free survival in resected acral lentiginous melanoma patients: A retrospective case series.

Adjuvant nivolumab therapy has been reported to improve the survival of melanoma patients. Acral lentiginous melanoma (ALM) has been reported to be less likely to respond to immune checkpoint inhibitors (ICIs) than other subtypes. However, the efficacy of adjuvant nivolumab therapy for ALM patients remains uncertain due to the low number of cases. In this single-center retrospective case series, we analyzed the clinical data of patients with resected stage III/IV ALM who were referred to our department between April 1, 2004 and March 31, 2022. The analyzed clinical data included age, sex, TNM stage, treatments, adverse events and disease-free survival (DFS). Enrolled patients were divided into a nivolumab group and a non-ICI group according to the adjuvant therapy they received. In total, 27 patients were included. The nivolumab and non-ICI groups had 5 and 22 patients, respectively. There were no significant differences in patient characteristics between the two groups. There were no serious treatment-related adverse events in the non-ICI group, but one patient in the nivolumab group developed type 1 diabetes. In the survival analysis, the DFS for the nivolumab group did not exceed that of the non-ICI group in postoperative adjuvant therapy for ALM patients. Given that adjuvant nivolumab therapy sometimes results in serious adverse effects, the administration of the therapy may need to be carefully considered, especially for ALM patients.

Fc-binding proteins enhance autoantibody-induced BP180 depletion in pemphigoid.

Immunoglobulins (Igs) consist of two antigen-binding regions (Fab) and one constant region (Fc). Protein A and protein G are bacterial proteins used for the purification of IgG by virtue of their high affinities for the Fc fragment. Rheumatoid factors are autoantibodies against IgG Fc fragments, which are present in the body under physiological conditions. Little is known about the influence of Fc-binding proteins on the pathogenicity of antibody-induced autoimmune diseases. Pemphigoid diseases are a group of autoimmune subepidermal blistering disorders that includes bullous pemphigoid and mucous membrane pemphigoid. IgGs targeting the non-collagenous NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) are known to induce skin fragility in mice and the depletion of BP180 in keratinocytes. In this study, mAb against NC16A in combination with Fc-binding proteins was found to enhance BP180 depletion. Although mAb against the C-terminus of BP180 does not show pathogenicity in vivo or in vitro, mAb treatment with Fc-binding proteins clearly induced skin fragility in mice and BP180 depletion in keratinocytes. Anti-BP180 mAbs and Fc-binding proteins were colocalized in the cytoplasm and at the basement membrane zone. Cell adhesion strengths were decreased in parallel with BP180 amounts. Clinically, bullous pemphigoid patients had higher rheumatoid factor titers than controls. Anti-BP180 mAb in combination with high-titer rheumatoid factor serum was found to enhance BP180 depletion. Furthermore, saliva from mucous membrane pemphigoid patients contained larger quantities of bacteria and Fc-binding proteins than controls. Our results suggest that Fc-binding proteins (rheumatoid factor or protein G) may enhance the pathogenicity of autoantibodies in pemphigoid diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Diagnosis and management of pemphigus: Recommendations of an international panel of experts.

BACKGROUND: Several European countries recently developed international diagnostic and management guidelines for pemphigus, which have been instrumental in the standardization of pemphigus management. OBJECTIVE: We now present results from a subsequent Delphi consensus to broaden the generalizability of the recommendations. METHODS: A preliminary survey, based on the European Dermatology Forum and the European Academy of Dermatology and Venereology guidelines, was sent to a panel of international experts to determine the level of consensus. The results were discussed at the International Bullous Diseases Consensus Group in March 2016 during the annual American Academy of Dermatology conference. Following the meeting, a second survey was sent to more experts to achieve greater international consensus. RESULTS: The 39 experts participated in the first round of the Delphi survey, and 54 experts from 21 countries completed the second round. The number of statements in the survey was reduced from 175 topics in Delphi I to 24 topics in Delphi II on the basis of Delphi results and meeting discussion. LIMITATIONS: Each recommendation represents the majority opinion and therefore may not reflect all possible treatment options available. CONCLUSIONS: We present here the recommendations resulting from this Delphi process. This international consensus includes intravenous CD20 inhibitors as a first-line therapy option for moderate-to-severe pemphigus.

The direct binding of collagen XVII and collagen IV is disrupted by pemphigoid autoantibodies.

The basement membrane zone (BMZ) is framed by hemidesmosomes and extracellular matrix (ECM) including collagen IV (COL4). Hemidesmosomes are multiprotein complexes that include collagen XVII (COL17). BMZ proteins can be targeted in autoimmune subepidermal blistering diseases, e.g., pemphigoid targeting COL17. The blistering mechanisms in pemphigoid have not been fully elucidated, especially in mucous membrane pemphigoid (MMP), which mainly affects the mucosa. In this study, we showed that oral lesions in pemphigoid may be attributed to the inhibition of protein-protein interactions by autoantibodies. Using immunoprecipitation, we revealed that COL17 directly binds to COL4 in normal human keratinocytes and normal human oral keratinocytes. In particular, the C-terminus of COL17 is binding site to COL4 in oral keratinocytes. The precise COL4-binding region on COL17 was determined by protein-protein binding assay to be from amino acid Gly1175 to Asp1340 on the C-terminus. MMP-IgG or mAb recognizing the C-terminus hindered the interaction of COL17 with COL4 in oral keratinocytes. Furthermore, keratinocyte adhesion strength to COL4-coated plates was significantly reduced by the treatment of mAb against the C-terminus. In addition, the inflammatory infiltrates around perilesions were significantly less in MMP compared to BP. These results indicate that pemphigoid IgG targeting the C-terminus plays a pathogenic role in blister formation in the oral mucosa to inhibit protein interactions with less inflammation.

Regulatory T cells in autoimmune skin diseases.

CD4+ Foxp3+ regulatory T cells (Tregs) are suppressors of immune activation and play a crucial role in the maintenance of peripheral tolerance. Mutations of Foxp3 result in fatal autoimmunity in multiple organs, including the skin, in both humans and mice. Many studies have demonstrated the altered frequency and functions of Tregs, changes in cytokine and chemokine levels related to Tregs and the differences in genetic background regarding Tregs in autoimmune skin disorders. Recent studies have extended our knowledge of certain properties of Tregs, especially skin-resident Tregs. In addition, some novel therapies have been performed by modulating the number and the function of Tregs. This review focuses on the role of Tregs in some autoimmune skin disorders, including alopecia areata, vitiligo, pemphigoid and pemphigus, and systemic sclerosis, and discusses questions that remain to be addressed.

The identification of autoantigens in mucous membrane pemphigoid using immortalized oral mucosal keratinocytes.

BACKGROUND: Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP. METHODS: Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients. RESULTS: There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/ß4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17. CONCLUSION: We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.

Regulatory T-cell dysfunction induces autoantibodies to bullous pemphigoid antigens in mice and human subjects.

BACKGROUND: Regulatory T (Treg) cells play a crucial role in peripheral immune tolerance in multiple organs, including the skin. Thus far, the effect of peripheral immune tolerance failure on autoantibody-related autoimmune reactions to the skin is unclear. OBJECTIVE: We sought to elucidate the target autoantigens in the skin under the condition of Treg cell dysfunction caused by forkhead box P3 (Foxp3) gene mutations in scurfy mice and patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. METHODS: Sera and skin from scurfy mice and sera from patients with IPEX syndrome were analyzed to detect target autoantigens by using immunofluorescence studies, ELISAs, and immunoblotting. The pathogenicity of scurfy IgG was examined by using a passive transfer experiment. CD4+ T cells from scurfy mice were transferred to immunodeficient mice to examine their pathogenicity. Signal transducer and activator of transcription 6 (Stat6)-/- scurfy mice were analyzed to further clarify the molecular pathway of autoantibody production. Follicular helper T-cell counts are measured in Stat6-/- scurfy mice and scurfy mice. RESULTS: Scurfy mice spontaneously generated IgG autoantibodies to the dermal-epidermal junction, which had been class-switched from IgM within 12 days after birth. The target autoantigens were murine BP230 and type XVII collagen (COL17). The scurfy polyclonal autoantibodies did not induce skin fragility in neonatal mice. Autoantibody production was induced by CD4+ T cells from scurfy mice and was ameliorated by Stat6 gene knockout in association with a decrease of follicular helper T cells. We also identified autoantibodies to COL17 and BP230 in patients with IPEX syndrome and found an association between production of autoantibodies to COL17 and an eczematous skin phenotype. CONCLUSIONS: Dysregulation of Treg cells generates autoantibodies to COL17 and BP230 in vivo.

γδ T Cells Protect the Liver and Lungs of Mice from Autoimmunity Induced by Scurfy Lymphocytes.

γδ T cells have been shown to have immunoregulatory functions in several experimental autoimmune models. A mutation of the Foxp3 gene leads to the absence of regulatory T cells (Tregs) and a fatal systemic autoimmune disease in scurfy mice. Transfer of scurfy lymphocytes to RAG deficient (RAG(-/-)) recipients reproduces the inflammatory phenotype of the scurfy donor, including hepatitis and pneumonitis. In this study, we show that TCRα(-/-) recipients, which lack αß T cells but have γδ T cells and B cells, are significantly protected from the hepatitis and pneumonitis, but not the dermatitis, induced by adoptive transfer of scurfy lymphocytes. Cotransfer of γδ T cells, but not B cells, prevented hepatitis and pneumonitis in RAG(-/-) recipients of scurfy lymphocytes. γδ T cells in the TCRα(-/-) recipients of scurfy cells markedly expanded and expressed a highly activated (CD62L(lo)CD44(hi)) phenotype. The activated γδ T cells expressed high levels of CD39 and NKG2D on their cell surface. A high frequency of scurfy T cells in TCRα(-/-) recipients produced IL-10, suggesting that γδ T cells may enhance suppressor cytokine production from scurfy T cells in TCRα(-/-) recipients. This study indicates that γδ T cells may contribute to the maintenance of immunological homeostasis by suppressing autoreactive T cells in liver and lung.