Vitiligo is an autoimmune disease in which the melanocytes die because of immune cytotoxicity or oxidative stress-induced cell death.1 Repigmentation can be achieved with immunosuppressive therapies, such as topical tacrolimus, topical ruxolitinib, and narrow-band UV therapies. However, the repigmentation often takes a long time, and only a minority of patients achieve satisfactory repigmentation. Therefore, there is a need to expand therapeutic options for this disease.

Vitiligo is an acquired depigmentation skin disease caused by immune-mediated death of melanocytes. The most common treatment for vitiligo is narrow band ultraviolet B phototherapy, which often is combined with topical therapies such as tacrolimus. However, patients’ responses to these treatments show large variations. To date, the mechanism for this heterogeneity is unknown, and there are no molecular indicators that can predict an individual patient’s response to therapy. The goal of this study is to identify clinical parameters and gene expression biomarkers associated with vitiligo response to therapy. Six patients with segmental vitiligo and 30 patients with non-segmental vitiligo underwent transcriptome sequencing of lesional and nonlesional skin at baseline before receiving combined UBUVB and tacrolimus therapy for 6 month, and were separated into good response and bad response groups based on target lesion achieving > 10% repigmentation or not. Our study revealed that treatment-responsive vitiligo lesions had significantly shorter disease duration compared with non-responsive vitiligo lesions (2.5 years vs 11.5 years, p=0.046, t-Test), while showing no significant differences in the age, gender, ethnicity, vitiligo subtype, or disease severity. Transcriptomic analyses identified a panel of 68 genes separating the good response from bad response lesions including upregulation of immune active genes, such as CXCL10, FCRL3, and TCR, Further, compared with vitiligo lesions with long disease duration, the lesions with short duration also have much higher level of expression of immune-active genes, including some (such as FCRL3 and TCR genes) that are associated with favorable therapeutic response. In conclusion, our study has identified clinical parameters such as short disease duration and a panel of immune active and other gene expression biomarkers that are associated with favorable response to immune suppressive NBUVB + tacrolimus therapy. These markers may be useful clinically for individualized therapeutic management of vitiligo patients in the future.

Lysyl oxidase-like 3 (LOXL3) is an extracellular enzyme involved in the synthesis of collagen and elastin, and it has been reported to promote melanoma cell proliferation and invasion in vitro. However, the expression level of LOXL3 at different stages of melanocytic lesions and the role of LOXL3 in melanoma pathogenesis remain unknown. Immunohistochemical staining of LOXL3 in a tissue microarray of 373 biopsies at different melanocytic stages was conducted. The correlation between LOXL3 expression and patient survival was examined using Kaplan–Meier survival analysis. Univariate and multivariate Cox regression analyses were conducted to study the hazard ratios. The tissue microarray study revealed that stronger expression of LOXL3 protein was found at more advanced melanocytic stages (P < 0.0001; χ 2 test). Increased LOXL3 expression was associated with enhanced tumor thickness and mitosis. Survival analysis showed significantly worsened prognosis in primary melanoma patients when the LOXL3 expression level was higher (P = 0.043; log-rank test). Multivariate Cox regression analysis further showed that LOXL3 expression is a prognostic factor for primary melanoma patient survival (P = 0.04). LOXL3 expression is positively correlated with tumor progression and invasion, and its overexpression is associated with worse prognosis of primary melanoma patients. LOXL3 can serve as a prognostic marker to help identify primary melanoma patients at higher risks of death.

Paget's disease (PD) is an intraepidermal adenocarcinoma of the skin at the breast (mammary PD) or urogenital locations (extramammary PD [EMPD]). At present, there is lack of clarity on PD's pathogenesis, the relationship between its subtypes, and its lineage link with the underlying invasive carcinomas. Here we describe that mammary PD and EMPD have similar mutational profiles, with the most frequent recurrent mutations occurring in the chromatin remodeling genes, such as KMT2C (MLL3, 39%) and ARID2 (22%), with additional recurrent somatic mutations detected in genes previously not known to be mutated in cancers, such as CDCC168 (34%), FSIP2 (29%), CASP8AP2 (29%), and BIRC6 (24%). In paired mammary PD and underlying breast carcinoma samples, distinct gene mutations were detected, indicating that they represent independent oncogenic events. Finally, multistage EMPD tissue sequencing revealed KMT2C gene occurring early in EMPD oncogenesis, and that multifocal EMPD samples share the same early gene mutations, suggesting clonal origin of multifocal EMPD. Our results reveal similar genomic landscapes between mammary PD and EMPD, including early aberrations in chromatin remodeling genes. In addition, mammary PD and underlying breast ductal carcinomas represent independent oncogenic events. These findings provide approaches for developing diagnostic tools and therapeutic interventions for PD.

Cutaneous T-cell lymphomas (CTCL) are the most common primary lymphomas of the skin. We have previously identified thymocyte selection-associated high mobility group (HMG) box protein (TOX) as a promising drug target in CTCL; however, there are currently no small molecules able to directly inhibit TOX. We aimed to address this unmet opportunity by developing anti-TOX therapeutics with the use of computer-aided drug discovery methods. The available NMR-resolved structure of the TOX protein was used to model its DNA-binding HMG-box domain. To investigate the druggability of the corresponding protein–DNA interface on TOX, we performed a pilot virtual screening of 200,000 small molecules using in silico docking and identified ‘hot spots’ for drug-binding on the HMG-box domain. We then performed a large-scale virtual screening of 7.6 million drug-like compounds that were available from the ZINC15 database. As a result, a total of 140 top candidate compounds were selected for subsequent in vitro validation. Of those, 18 small molecules have been characterized as selective TOX inhibitors.

Paget's disease (PD) is an uncommon intraepithelial adenocarcinoma with unknown pathogenesis. There are two anatomic subtypes: mammary (MPD) and extramammary (EMPD). Little is known about their molecular characteristics. Our objective was to discover novel molecular markers for PD and its subtypes. In the discovery phase, we used transcriptome analyses to uncover the most differentially expressed genes and pathways in EMPD biopsies compared with normal skin. In the validation phase, we performed immunohistochemistry analyses on the most promising marker (FOXA1) and other markers selected from a literature review (GATA3, estrogen receptor [ER], and androgen receptor [AR]) on independent biopsies of MPD (n = 86), EMPD (n = 59), and normal skin (n = 21). Transcriptome analyses revealed 210 genes differentially expressed more than 10-fold between EMPD and normal skin. These genes are involved in mammary and sweat gland development (FOXA1) and immune regulation, as well as epidermal differentiation. Immunohistochemistry staining revealed that FOXA1 was positive in 88% of both MPD and EMPD, whereas GATA3 was positive in 67% of MPD and 77% of EMPD, and ER was positive in 9% of MPD and 19% of EMPD. Finally, AR was positive in 33% of PD and 54% of EMPD. Mammary Paget's disease and EMPD share dysregulation of the glandular developmental regulator gene FOXA1, suggesting similarity in cell-specific transcriptional regulation. Further, FOXA1 may be a useful molecular target for developing PD therapies.

BackgroundThere is a wide variation in the reported prevalence of primary hyperhidrosis in the literature. Further, it is unknown if primary hyperhidrosis is a lifelong condition, or if demographical factors influence hyperhidrosis prevalence.ObjectivesThis study aims to examine the prevalence of hyperhidrosis in multiple ethnic groups from two ethnically diverse cities and to determine if the prevalence of primary hyperhidrosis changes according to age, gender, ethnicity, body mass index, and geographical locations.MethodsIn total, 1010 consecutive subjects attending dermatology outpatient clinics in Shanghai Skin Disease Hospital and 1018 subjects in Skin Care Center of Vancouver General Hospital were invited to fill out a questionnaire on their presenting concerns, demographical information, and sweating symptoms. The subjects were then classified to have primary hyperhidrosis using the criteria of International Hyperhidrosis Society, late-onset hyperhidrosis, or no-hyperhidrosis. The prevalence of primary HH and late-onset HH was calculated for the entire study population and in subgroups stratified according to age of examination, sex, ethnicity, presenting diagnosis, bodymass index, and specific study cities. Multivariate logistic regression analyses were performed to assess the impact of these factors on HH prevalence.ResultsThe prevalence of primary hyperhidrosis is very similar in Shanghai and in Vancouver, at 14.5% and 12.3% respectively. In addition, 4.0% of subjects in Shanghai and 4.4% subjects in Vancouver suffer from late-onset HH. Primary HH has highest prevalence in those younger than 30 years of age, decreasing dramatically in later years. Caucasian subjects are at least 2.5 times more likely to develop axillary hyperhidrosis compared to Chinese subjects. Obesity does not have much influence on primary HH presentation, although it does increase significantly the development of late-onset HH. Finally, there is no major difference of hyperhidrosis between Chinese subjects in Shanghai and Vancouver.LimitationsThe data were gathered according to patients' self-reports only and the sample size was relatively small in some groups after stratification for gender, ethnicity and age.ConclusionPrevalence of primary HH and late-onset HH is similar in dermatology outpatients independent of geographical locations. However, certain specific HH subtypes can show great variations according to ethnicity, age, body mass index and sex.

Background: There are conflicting data about the correlation between hyperhidrosis (HH) and anxiety and depression.Objective: We sought to determine the prevalence of anxiety and depression in patients with or without HH.Methods: We examined 2017 consecutive dermatology outpatients from Vancouver, British Columbia, Canada, and Shanghai, China, using Patient Health Questionnaire-9 and Generalized Anxiety Disorder-7 scales for anxiety and depression assessments. Multivariable logistic regression analysis was performed to evaluate if the impact of HH on anxiety and depression is dependent on demographic factors and diagnoses of the patients' presenting skin conditions.Results: The prevalence of anxiety and depression was 21.3% and 27.2% in patients with HH, respectively, and 7.5% and 9.7% in patients without HH, respectively (P value <.001 for both). There were positive correlations between HH severity and the prevalence of anxiety and depression. Multivariable analysis showed that HH-associated increase in anxiety and depression prevalence is independent of demographic factors and presenting skin conditions.Limitation: The data from the questionnaires relied on the accuracy of patients' self-reports.Conclusion: Both single variant and multivariable analyses showed a significant association between HH and the prevalence of anxiety and depression in a HH severity dependent manner.

Hypertrophic scars and keloids are devastating fibrotic conditions. Despite advances in knowledge and various therapeutic methods prevention and treatment of these conditions remains a challenge. Our group has previously shown that kynurenic acid (KyA) as a topical formulation reduces hypertrophic scarring in rabbit ear model. In this study we hypothesized that the use of a biocompatible and biodegradable polymer microsphere for controlled slow release of KyA will reduce fibrosis in closed wound in a rat model. The FDA approved Poly (lactic-co-glycolic acid) (PLGA) polymer was used to encapsulate KyA. An animal model of wound healing which involves subcutaneous implantation of pre-cut PVA sponges in rat was used to evaluate the in vivo efficacy of the microspheres. The in vitro experiments revealed a successful encapsulation of KyA(average encapsulation efficiency=80.65%±18.49) and a release profile that showed a gradual release over 35 days following a lag phase for 30 days. Both histological examination and hydroxyproline assay of the samples harvested after 66 days revealed a significant reduction in collagen deposition inside and around the PVA sponge implants loaded with KyA microspheres compared to the PVA alone or loaded with empty microspheres (0.3±0.5, 6.74± 2.77, 2.7±o.89 mg collagen/PVA respectively). There was no significant difference between samples collected after 35 days. Our data suggests that gradual release of KyA after 30 days can prevent fibrosis in vivo while the lag phase allows normal healing process to occur. This drug delivery system provides a novel approach toward prevention of fibrosis after surgical interventions. Category: Applied/functional experiments VERTICAL SECTIONING MULTIMODALITY VIDEO MICROSCOPY OF HUMAN SKIN

1The Department of Dermatology and Skin Science, University of British Columbia and Pacific Dermaesthetics, Vancouver, British Columbia. 2Faculty of Medicine, University of British Columbia, Vancouver, British Columbia. 3DynalifeDx, University of Alberta, Departments of Pathology and Dermatology, University of Alberta, Edmonton, Alberta. 4Department of Medicine (Dermatology), University of Toronto, Toronto, Ontario

Abstract INTRODUCTION: SUM-6 is a human homeobox gene that encodes for a transcription factor, which plays a key role in normal embryogenesis. Overexpression of both the gene and nuclear protein has been shown to occur in a variety of cancers such as breast, colorectal, and pancreatic, and has been demonstrated to promote tumorigenesis. The purpose of the current study was to evaluate the expression of the SUM-6 protein, its clinical relevance, and biological function in melanoma. EXPERIMENTAL PROCEDURES: Immunohistochemistry using a specific SUM-6 antibody was performed on a tissue microarray consisting of 438 patient biopsies, which included benign and malignant melanocytic tumors. Double-blinded scoring of distinct nuclear and cytoplasmic SUM-6 staining was performed. SPSS 16.0 statistical package and Microsoft Excel were used to carry out the analyses of data. Analyses included the χ2 test and univariate analysis by the log-rank test in conjunction with the Kaplan-Meier survival curve for visualization. RESULTS: Nuclear staining of SUM-6 was detected in 100% of normal nevi (n = 19), 81% of dysplastic nevi (n = 32), 87% of melanoma in situ (n = 23), 79% of primary melanoma (n = 219), and 53% of metastatic melanoma (n = 145), whereas cytoplasmic staining was detected in 63%, 94%, 56%, 94%, and 97%, respectively. Univariate analysis revealed that five year disease-specific survival decreased significantly when SUM-6 was excluded from the nucleus and when SUM-6 levels were elevated in the cytoplasm (p = 0.001 and 0.019, respectively). CONCLUSIONS: We concluded that melanoma progression and metastasis is associated with a profound shift of transcription factor SUM-6 from the nucleus to the cytoplasm. It is interesting to note that in most other cancers, the SUM-6 protein was instead overexpressed in the nucleus. The patients in whom SUM-6 was excluded from the nucleus demonstrated a significantly worse prognosis than the patients who retained nuclear expression. These findings suggest that either the absence of nuclear expression or elevated cytoplasmic presence (or both) of SUM-6 may play a role in the progression of malignant melanoma. Citation Format: Laura J. Graziano, Xue Zhang, Yabin Cheng, Gang Wang, Mingwan Su, Magdalena Martinka, Youwen Zhou. Melanoma progression involves a profound nuclear to cytoplasmic shift of the transcription factor SUM-6 (specifically upregulated in melanoma gene six). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5031.

Mycosis fungoides (MF) often mimics the common chronic inflammatory skin diseases and is difficult to be diagnosed with certainty, partly because of the lack of well-characterized molecular markers. Previously, we discovered that TOX, a key T cell development regulator,was aberrantly over-expressed in early stage MF. In the current multi-center study involving two independent patient cohorts, we determined the prevalence of TOX over-expression in the full spectrum of MF skin biopsies, and tested if TOX expression levels correlated with long term clinical outcomes. We examined TOX expression levels in 113 MF biopsies. We found that the MF biopsies expressed higher TOX mRNA than the controls in both cohorts (17.9 fold in cohort 1, P = 0.002; 5.8 fold in cohort 2, P < 0.0001). In addition, thicker skin lesions such as plaques and tumors expressed even higher TOX levels than thinner patches. Further, TOX over-expression differentiated MF from the controls (area under the curve [AUC]=0.87, P < 0.0001). Finally, high TOX mRNA levels correlated with increased risks of disease progression (P = 0.003) and disease-specific mortality (P = 0.008). In conclusion, TOX may be a useful marker for improving MF diagnosis and prognostication.

Neutralizing CXCL10 reverses established vitiligo.

TOX is a nuclear factor essential for the development of CD4(+) T cells in the thymus. It is normally expressed in low amounts in mature CD4(+) T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), as compared to normal skin or benign inflammatory dermatoses. However, its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study, we demonstrate that TOX expression is also enhanced significantly in primary CD4(+)CD7(-) cells from patients with Sézary syndrome, a leukemic variant of CTCL, and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression, leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore, TOX knockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors, CDKN1B and CDKN1C. Lastly, blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively, these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL.

To identify susceptibility loci for vitiligo, we extended our previous vitiligo genome-wide association study with a two-staged replication study that included 6,857 cases and 12,025 controls from the Chinese Han population. We identified three susceptibility loci, 12q13.2 (rs10876864, P-combined = 8.07 X 10(-12), odds ratio (OR) = 1.18), 11q23.3 (rs638893, P-combined = 2.47 x 10(-9), OR = 1.22), and 10q22.1 (rs1417210, P-combined = 1.83 x 10(-8), OR = 0.88), and confirmed three previously reported loci for vitiligo, 3q28 (rs9851967, P-combined = 8.57 x 10(-8), OR = 0.88), 10p15.1 (rs3134883, P-combined = 1.01 x 10(-5), OR = 1.11), and 22q12.3 (rs2051582, P-combined = 2.12 x 10(-5), OR = 1.14), in the Chinese Han population. The most significant single-nucleotide polymorphism in the 12q13.2 locus is located immediately upstream of the promoter region of PMEL, which encodes a major melanocyte antigen and has expression loss in the vitiligo lesional skin. In addition, both 12q13.2 and 11q23.3 loci identified in this study are also associated with other autoimmune diseases such as type 1 diabetes and systemic lupus erythematosus. These findings provide indirect support that vitiligo pathogenesis involves a complex interplay between immune regulatory factors and melanocyte-specific factors. They also highlight similarities and differences in the genetic basis of vitiligo in Chinese and Caucasian populations. Journal of Investigative Dermatology (2013) 133, 403-410; doi:10.1038/jid.2012.320; published online 6 September 2012

The lack of a specific marker differentiating early mycosis fungoides (eMF) from benign inflammatory dermatitis presents significant difficulties in the assessment and management of suspected MF patients, which often leads to delayed diagnosis and improper medical approaches. To address this, an investigation was carried out to characterize positive identification markers for eMF by comparing eMF lesions with healthy skin and benign inflammatory dermatitis, using high-throughput genomic transcription profiling. A total of 349 genes were differentially expressed in eMF lesions compared with normal skin. These genes belong to pathways associated with inflammation, immune activation, and apoptosis regulation. Most of them (N=330) also demonstrated significant upregulation in chronic dermatitis, making them nonideal markers for eMF. Among them, 19 genes with specific enrichment in eMF lesions were identified that showed no significant upregulation in chronic dermatitis. Two of them, TOX and PDCD1, showed high discrimination power between eMF lesions and biopsies from benign dermatitis by RNA expression. Furthermore, TOX demonstrated highly specific staining of MF cells in eMF skin biopsies in immunohistochemistry and immunofluorescence, including the early epidermotropic cells in Pautrier's microabscesses. This study demonstrates the potential of eMF-enriched genes, especially TOX, as molecular markers for histological diagnosis of eMF, which currently is a major diagnostic challenge.

BACKGROUND: Vitiligo is characterized by the death of melanocytes in the skin. This is associated with the presence of T cell infiltrates in the lesional borders. However, at present, there is no detailed and systematic characterization on whether additional cellular or molecular changes are present inside vitiligo lesions. Further, it is unknown if the normal appearing non-lesional skin of vitiligo patients is in fact normal. The purpose of this study is to systematically characterize the molecular and cellular characteristics of the lesional and non-lesional skin of vitiligo patients. METHODS AND MATERIALS: Paired lesional and non-lesional skin biopsies from twenty-three vitiligo patients and normal skin biopsies from sixteen healthy volunteers were obtained with informed consent. The following aspects were analyzed: (1) transcriptome changes present in vitiligo skin using DNA microarrays and qRT-PCR; (2) abnormal cellular infiltrates in vitiligo skin explant cultures using flow cytometry; and (3) distribution of the abnormal cellular infiltrates in vitiligo skin using immunofluorescence microscopy. RESULTS: Compared with normal skin, vitiligo lesional skin contained 17 genes (mostly melanocyte-specific genes) whose expression was decreased or absent. In contrast, the relative expression of 13 genes was up-regulated. The up-regulated genes point to aberrant activity of the innate immune system, especially natural killer cells in vitiligo. Strikingly, the markers of heightened innate immune responses were also found to be up-regulated in the non-lesional skin of vitiligo patients. CONCLUSIONS AND CLINICAL IMPLICATIONS: As the first systematic transcriptome characterization of the skin in vitiligo patients, this study revealed previously unknown molecular markers that strongly suggest aberrant innate immune activation in the microenvironment of vitiligo skin. Since these changes involve both lesional and non-lesional skin, our results suggest that therapies targeting the entire skin surface may improve treatment outcomes. Finally, this study revealed novel mediators that may facilitate future development of vitiligo therapies.