Comparative Quantitative Proteomic Analysis of Melanoma Subtypes, Nevus-Associated Melanoma, and Corresponding Nevi.

A substantial part of cutaneous malignant melanomas develops from benign nevi. However, the precise molecular events driving the transformation from benign to malignant melanoma are not well-understood. We used laser microdissection and mass spectrometry to analyze the proteomes of melanoma subtypes, including superficial spreading melanomas (n = 17), nodular melanomas (n = 17), and acral melanomas (n = 15). Furthermore, we compared the proteomes of nevi cells with those of melanoma cells within the same specimens (nevus-associated melanoma (n = 14)). In total, we quantified 7935 proteins. Despite the genomic and clinical differences of the melanoma subtypes, our analysis revealed relatively similar proteomes, except for the upregulation of proteins involved in immune activation in nodular melanomas versus acral melanomas. Examining nevus-associated melanoma versus nevi, we found 1725 differentially expressed proteins (false discovery rate < 0.05). Among these proteins were 140 that overlapped with cancer hallmarks, tumor suppressors, and regulators of metabolism and cell cycle. Pathway analysis indicated aberrant activation of the phosphoinositide 3-kinase-protein kinase B-mTOR pathways and the Hippo-YAP pathway. Using a classifier, we identified six proteins capable of distinguishing melanoma from nevi samples. Our study represents a comprehensive comparative analysis of the proteome in melanoma subtypes and associated nevi, offering insights into the biological behavior of these distinct entities.

LAG3 and TIGIT Expression on Tumor-Infiltrating Lymphocytes in Cutaneous Melanoma.

BACKGROUND: Melanoma is widely recognized to be an immunogenic tumor that often contains tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment. During cancer progression, expression of ligands that bind immune checkpoint (IC) proteins, such as PD-1, expressed on the surface of TILs, hinder them from exerting their antitumor functions. TILs consist of a heterogenous group of immune cells and their presence is associated with an improved overall survival in melanoma patients. Introduction of IC inhibitors has revolutionized management and prognosis of advanced melanoma. Unfortunately, the response rates have continued to be limited, resulting in growing interest in characterizing novel IC proteins, and developing combination therapy that includes inhibitors against multiple IC proteins. METHODS: In a regional cohort of 166 patients diagnosed with cutaneous superficial spreading melanoma with different degree of TILs, we investigated the tumor immune-associated gene expression profile using NanoString Technology. We used multiplex immunofluorescence (mIF) staining in a subset of tumors (N = 7), combining IC proteins T-cell immunoglobulin and ITIM domain (TIGIT) and LAG3 with a melanoma cell marker (SOX10) and immune cell markers (CD8 [cytotoxic T cells], CD4 [T helper cells], FOXP3 [regulatory T cells/Tregs], PAX5 [B cells], and CD56 [NK/NKT cells]) and IC protein PD-1. RESULTS: We found upregulation of 91 differentially expressed genes, including IC proteins, LAG3 and TIGIT in melanomas with brisk TILs compared to tumors where TILs were absent. mIF staining revealed LAG3 and TIGIT expression in the majority of CD8+ T cells. Only few Tregs and CD4+ T cells expressed LAG3, whereas majority of them expressed TIGIT. LAG3 and TIGIT were expressed in a small fraction of the NK/NKT cells and lacked in the B cells. The majority of PD-1+ cells co-localized with LAG3 and TIGIT. CONCLUSION: We report a variable expression of LAG3 and TIGIT on TILs subtypes and a coeval occurrence with PD-1. This knowledge places LAG3 and TIGIT in spatial and cellular context in melanoma. The data suggest that targeting multiple IC proteins might help overcome the current challenges with IC therapies.

The Proteome of Hand Eczema Assessed by Tape Stripping.

Hand eczema (HE) is a prevalent skin disease. However, the classification of HE into different subtypes remains challenging. A limited number of previous studies have employed invasive biopsy-based strategies; yet, studies of the HE proteome using noninvasive tape-stripping methodology have not been reported. In this study, we wanted to assess whether global proteomic analysis of skin tape strip samples can be used for subclassification of patients with HE. Tape strips were collected from patients with HE and healthy skin. Liquid chromatography-mass spectrometry proteomics was performed, and the global protein expression was analyzed. We identified 2,919 proteins in stratum corneum-derived skin cells from tape strip samples. Compared with healthy skin, the lesional samples from patients with HE exhibited increased expression of immune-related markers and a decreased expression of structural barrier proteins. The difference between HE subtypes was restricted to the lesional skin areas and included an increased expression of skin barrier-related proteins independently of the concurrent AD. In conclusion, we found that the noninvasive tape strip method used in combination with liquid chromatography-mass spectrometry proteomics can be used for analysis of skin protein expression in patients with HE. Thus, the method shows potential for assessing the proteomic differences between subtypes of HE and biomarker discovery.

BRAFV600E Expression Is Homogenous and Associated with Nonrecurrent Disease and Better Survival in Primary Melanoma.

BACKGROUND: Superficial spreading melanomas (SSMs) are the most common type of melanoma and cause the majority of skin cancer deaths. More than 50% of cases harbor a mutation in the BRAF gene that activates the mitogen-activated protein kinase (MAPK) cancer signaling pathway. BRAFV600E is the most common BRAF mutation, and it represents an important biomarker that guides treatment selection. However, the relationship between the BRAFV600E gene expression and intratumoral protein distribution, on one side, and clinicopathological factors and patient outcomes, on the other, is not fully described. Additionally, whether MAPK cancer signaling activation in melanoma is due to increased biochemical activity of BRAFV600E, increased mRNA levels, or both requires further investigation. Here, we addressed these questions by examining expression patterns of BRAFV600E in primary treatment-naive melanomas and correlating them to clinicopathological factors and patient outcomes. METHODS: In 166 SSM cases, we performed immunohistochemical staining to investigate the protein expression of BRAFV600E, and we measured BRAF mRNA levels using NanoString nCounter system. RESULTS: Ninety-seven (49%) melanomas stained positive for BRAFV600E, with nearly 100% intratumoral homogeneity observed. Positive BRAFV600E expression was significantly associated with nonrecurrent disease and was found to be an independent predictor of better prognosis in univariate and multivariable analyses. Furthermore, presence of tumor-infiltrating lymphocytes, sentinel lymph node biopsy negativity, and low Breslow thickness were all independent predictors of better prognosis. We observed no difference in the BRAF mRNA levels in BRAFV600E-negative and BRAFV600E-positive melanomas, respectively. Validation in a larger publicly available cohort confirmed that there is only a weak correlation (Spearman 0.4) between BRAFV600E mRNA and protein levels and no differences in mRNA between BRAFV600E mutated and non-mutated patients. CONCLUSION: Our findings indicated that BRAFV600E is homogeneously present throughout the whole tumor and is associated with nonrecurrent disease and better survival in primary melanoma. We also showed that BRAFV600E mutation does not result in higher transcriptional levels, suggesting that activation of the MAPK signaling pathway in BRAFV600E mutated patients can be attributed to the increased biochemical activity caused by the mutation.

Deep Visual Proteomics defines single-cell identity and heterogeneity.

Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.

Towards Precision Dermatology: Emerging Role of Proteomic Analysis of the Skin.

BACKGROUND: The skin is the largest organ in the human body and serves as a multilayered protective shield from the environment as well as a sensor and thermal regulator. However, despite its importance, many details about skin structure and function at the molecular level remain incompletely understood. Recent advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics have enabled the quantification and characterization of the proteomes of a number of clinical samples, including normal and diseased skin. SUMMARY: Here, we review the current state of the art in proteomic analysis of the skin. We provide a brief overview of the technique and skin sample collection methodologies as well as a number of recent examples to illustrate the utility of this strategy for advancing a broader understanding of the pathology of diseases as well as new therapeutic options. KEY MESSAGES: Proteomic studies of healthy skin and skin diseases can identify potential molecular biomarkers for improved diagnosis and patient stratification as well as potential targets for drug development. Collectively, efforts such as the Human Skinatlas offer improved opportunities for enhancing clinical practice and patient outcomes.

Immunoregulatory and lipid presentation pathways are upregulated in human face transplant rejection.

BACKGROUNDRejection is the primary barrier to broader implementation of vascularized composite allografts (VCAs), including face and limb transplants. The immunologic pathways activated in face transplant rejection have not been fully characterized.METHODSUsing skin biopsies prospectively collected over 9 years from 7 face transplant patients, we studied rejection by gene expression profiling, histology, immunostaining, and T cell receptor sequencing.RESULTSGrade 1 rejection did not differ significantly from nonrejection, suggesting that it does not represent a pathologic state. In grade 2, there was a balanced upregulation of both proinflammatory T cell activation pathways and antiinflammatory checkpoint and immunomodulatory pathways, with a net result of no tissue injury. In grade 3, IFN-γ-driven inflammation, antigen-presenting cell activation, and infiltration of the skin by proliferative T cells bearing markers of antigen-specific activation and cytotoxicity tipped the balance toward tissue injury. Rejection of VCAs and solid organ transplants had both distinct and common features. VCA rejection was uniquely associated with upregulation of immunoregulatory genes, including SOCS1; induction of lipid antigen-presenting CD1 proteins; and infiltration by T cells predicted to recognize CD1b and CD1c.CONCLUSIONOur findings suggest that the distinct features of VCA rejection reflect the unique immunobiology of skin and that enhancing cutaneous immunoregulatory networks may be a useful strategy in combatting rejection.Trial registrationClinicalTrials.gov NCT01281267.FUNDINGAssistant Secretary of Defense and Health Affairs, through Reconstructive Transplant Research (W81XWH-17-1-0278, W81XWH-16-1-0647, W81XWH-16-1-0689, W81XWH-18-1-0784, W81XWH-1-810798); American Society of Transplantation's Transplantation and Immunology Research Network Fellowship Research Grant; Plastic Surgery Foundation Fellowship from the American Society of Plastic Surgeons; Novo Nordisk Foundation (NNF15OC0014092); Lundbeck Foundation; Aage Bangs Foundation; A.P. Moller Foundation for the Advancement of Medical Science; NIH UL1 RR025758.

Molecular analysis of primary melanoma T cells identifies patients at risk for metastatic recurrence.

Primary melanomas >1 mm thickness are potentially curable by resection, but can recur metastatically. We assessed the prognostic value of T cell fraction (TCFr) and repertoire T cell clonality, measured by high-throughput-sequencing of the T cell receptor beta chain (TRB) in T2-T4 primary melanomas (n=199). TCFr accurately predicted progression-free survival (PFS) and was independent of thickness, ulceration, mitotic rate, or age. TCFr was second only to tumor thickness in its predictive value, using a gradient boosted model. For accurate PFS prediction, adding TCFr to tumor thickness was superior to adding any other histopathological variable. Furthermore, a TCFr >20% was protective regardless of tumor ulceration status, mitotic rate or presence of nodal disease. TCFr is a quantitative molecular assessment that predicts metastatic recurrence in primary melanoma patients whose disease has been resected surgically. This study suggests that a successful T cell-mediated antitumor response can be present in primary melanomas.

NKG2D-dependent activation of dendritic epidermal T cells in contact hypersensitivity.

The interaction between keratinocytes (KCs) and skin-resident immune cells has an important role in induction of contact hypersensitivity. A specific subset of γδ T cells termed dendritic epidermal T cells (DETCs) are located in mouse epidermis, and we have recently shown that DETCs become activated and produce IL-17 in an IL-1ß-dependent manner during contact hypersensitivity. Various receptors on DETCs, including NKG2D, are involved in DETC responses against tumors and during wound healing. The ligands for NKG2D (NKG2DL) are stress-induced proteins such as mouse UL16-binding protein-like transcript 1 (Mult-1), histocompatibility 60 (H60), and retinoic acid early inducible-1 (Rae-1) in mice and major histocompatibility complex (MHC) class I-chain-related A (MICA), MHC class I-chain-related B, and UL16-binding protein in humans. Here, we show that allergens upregulate expression of the NKG2DL Mult-1, H60, and Rae-1 in cultured mouse KCs and of MICA in primary human KCs. We demonstrate that Mult-1 is expressed in mouse skin exposed to allergen. Furthermore, we find that the vast majority of DETCs in murine epidermis and skin-homing cutaneous lymphocyte-associated antigen positive γδ T cells in humans express NKG2D. Finally, we demonstrate that blocking of NKG2D partially inhibits allergen-induced DETC activation. These findings demonstrate that NKG2D and NKG2DL are involved in allergen-induced activation of DETCs and indicate that the NKG2D/NKG2DL pathway might be a potential target for treatment of contact hypersensitivity.

Epicutaneous exposure to nickel induces nickel allergy in mice via a MyD88-dependent and interleukin-1-dependent pathway.

BACKGROUND: Several attempts to establish a model in mice that reflects nickel allergy in humans have been made. Most models use intradermal injection of nickel in combination with adjuvant to induce nickel allergy. However, such models poorly reflect induction of nickel allergy following long-lasting epicutaneous exposure to nickel. OBJECTIVE: To develop a mouse model reflecting nickel allergy in humans induced by epicutaneous exposure to nickel, and to investigate the mechanisms involved in such allergic responses. METHODS: Mice were exposed to NiCl2 on the dorsal side of the ears. Inflammation was evaluated by the swelling and cell infiltration of the ears. T cell responses were determined as numbers of CD4+ and CD8+ T cells in the draining lymph nodes. Localization of nickel was examined by dimethylglyoxime staining. RESULTS: Epicutaneous exposure to nickel results in prolonged localization of nickel in the epidermis, and induces nickel allergy in mice. The allergic response to nickel following epicutaneous exposure is MyD88-dependent and interleukin (IL)-1 receptor-dependent, but independent of toll-like receptor (TLR)-4. CONCLUSION: This new model for nickel allergy that reflects epicutaneous exposure to nickel in humans shows that nickel allergy is dependent on MyD88 and IL-1 receptor signalling, but independent of TLR4.

CD4(+) T cells producing interleukin (IL)-17, IL-22 and interferon-γ are major effector T cells in nickel allergy.

BACKGROUND: It has been suggested that interleukin (IL)-17 and IL-22 play important roles in the elicitation of human allergic contact dermatitis; however, the frequencies of T cell subtypes producing IL-17 and IL-22 in human allergic contact dermatitis are unknown. OBJECTIVES: To determine the frequencies of CD4(+) , CD8(+) and γδ T cells producing IL-17, IL-22 and interferon (IFN)-γ in the blood and skin from nickel-allergic patients. PATIENTS/MATERIALS/METHODS: Blood samples were collected from 14 patients and 17 controls, and analysed by flow cytometry. Biopsies were taken from 5 patients and 6 controls, and analysed by immunohistochemistry and flow cytometry of skin lymphocytes. RESULTS: We found an increased frequency of γδ T cells in the blood, but no differences in the distribution of cytokine-producing CLA(+) T cell subtypes in nickel-allergic patients as compared with controls. In nickel-allergic patients, there was massive cellular infiltration dominated by CD4(+) T cells producing IL-17, IL-22 and IFN-γ in nickel-challenged skin but not in vehicle-challenged skin. CONCLUSION: CD4(+) T cells producing IL-17, IL-22 and IFN-γ are important effector cells in the eczematous reactions of nickel-induced allergic contact dermatitis in humans.

Patients newly diagnosed with clinical type 2 diabetes during oral glucocorticoid treatment and observed for 14 years: all-cause mortality and clinical developments.

Chronic exposure to glucocorticoids (GCs) has many side effects including glucose intolerance and diabetes and may accelerate the occurrence of cardiovascular disease and increase mortality. We studied the 14-year clinical development of diabetes in patients diagnosed with diabetes during GC treatment. A population-based sample of 1369 people newly diagnosed with clinical type 2 diabetes underwent a clinical examination at diagnosis, and surviving patients were followed up 6 and 14 years later. Patients receiving oral GC treatment at diagnosis were compared with the other patients. Of 1369 patients, 35 (2.6%) were treated with oral GCs at diabetes diagnosis. At that point, patients on GC therapy were older (69.9 versus 65.3 years, p = 0.007, sex-adjusted) and tended to have lower BMI (26.1 versus 29.1 kg/m(2) , p = 0.023), also 6 years after diagnosis (24.8 versus 28.4, p = 0.011), than patients not being treated with GCs. In a univariate Cox regression model, GC treatment at diagnosis increased all-cause mortality with a hazard ratio (95% confidence interval) of 2.01 (1.39-2.89, p = 0.0002, n = 1369), while this decreased to 1.41 (0.98-2.04, p = 0.065, n = 1369) when adjusted for age and sex and to 1.39 (0.92-2.11, p = 0.12, n = 1086) when risk factors, complications and cancer were added to the model. Apart from differences in age and overweight, patients in this relatively small sample of those diagnosed with clinical type 2 diabetes during GC treatment were comparable at diagnosis and during 14 years of follow-up with those not treated with GCs, including with regard to the adjusted mortality rate.