Senescent human melanocytes drive skin ageing via paracrine telomere dysfunction.

Cellular senescence has been shown to contribute to skin ageing. However, the role of melanocytes in the process is understudied. Our data show that melanocytes are the only epidermal cell type to express the senescence marker p16INK4A during human skin ageing. Aged melanocytes also display additional markers of senescence such as reduced HMGB1 and dysfunctional telomeres, without detectable telomere shortening. Additionally, senescent melanocyte SASP induces telomere dysfunction in paracrine manner and limits proliferation of surrounding cells via activation of CXCR3-dependent mitochondrial ROS. Finally, senescent melanocytes impair basal keratinocyte proliferation and contribute to epidermal atrophy in vitro using 3D human epidermal equivalents. Crucially, clearance of senescent melanocytes using the senolytic drug ABT737 or treatment with mitochondria-targeted antioxidant MitoQ suppressed this effect. In conclusion, our study provides proof-of-concept evidence that senescent melanocytes affect keratinocyte function and act as drivers of human skin ageing.

Differential proteomic expression in indolent versus transforming oral lichen planus.

Oral lichen planus (OLP) confers an approximately 1% risk of transformation to oral squamous cell carcinoma (OSCC). Early identification of high-risk OLP would be very helpful for optimal patient management. We aimed to discover specific tissue-based protein biomarkers in patients with OLP who developed OSCC compared to those who did not. We used laser capture microdissection- and nanoLC-tandem mass spectrometry to assess protein expression in fixed lesional mucosal specimens in patients with indolent OLP (no OSCC after at least 5-year follow-up, n = 6), transforming OLP (non-dysplastic epithelium with lichenoid inflammation marginal to OSCC, n = 6) or normal oral mucosa (NOM, n = 5). Transforming OLP protein profile was enriched for actin cytoskeleton, mitochondrial dysfunction and oxidative phosphorylation pathways. CA1, TNNT3, SYNM and MB were overexpressed, and FBLN1 was underexpressed in transforming OLP compared with indolent OLP. Integrin signalling and antigen presentation pathways were enriched in both indolent and transforming OLP compared with NOM. This proteomic study provides potential biomarkers, such as CA1 overexpression, for higher-risk OLP. While further validation studies are needed, we propose that epithelial-mesenchymal transition may be involved in OLP carcinogenesis.

Differential proteomic expression profiles in vulvar lichen planus as compared to normal vulvar tissue, vulvar lichen sclerosus, or oral lichen planus: An exploratory study.

Vulvar lichen planus (VLP) is a chronic inflammatory disease which adversely affects patients' quality of life. The pathogenesis of VLP is unknown although Th1 immune response has been implicated. We aimed to discover specific tissue-based protein biomarkers in VLP compared to normal vulvar tissue (NVT), vulvar lichen sclerosus (VLS) and oral lichen planus (OLP). We used laser capture microdissection-liquid chromatography- tandem mass spectrometry to assess protein expression in fixed lesional mucosal specimens from patients with VLP (n = 5). We then compared proteomic profiles against those of NVT (n = 4), VLS (n = 5), OLP (n = 6) and normal oral mucosa (n = 5), previously published by our group. IL16, PTPRC, PTPRCAP, TAP1 and ITGB2 and were significantly overexpressed in VLP compared to NVT. Ingenuity pathway analysis identified antigen presentation and integrin signalling pathways. Proteins overexpressed in both VLP versus NVT and OLP versus NOM included IL16, PTPRC, PTPRCAP, TAP1, HLA-DPB1, HLA-B and HLA-DRA. This proteomic analysis revealed several overexpressed proteins in VLP that relate to Th1 autoimmunity, including IL16. Overlapping pathways, including those involving IFNγ and Th1 signalling, were observed between VLP, VLS, and OLP.

Rabgap1 promotes recycling of active ß1 integrins to support effective cell migration.

Integrin function depends on the continuous internalization of integrins and their subsequent endosomal recycling to the plasma membrane to drive adhesion dynamics, cell migration and invasion. Here we assign a pivotal role for Rabgap1 (GAPCenA) in the recycling of endocytosed active ß1 integrins to the plasma membrane. The phosphotyrosine-binding (PTB) domain of Rabgap1 binds to the membrane-proximal NPxY motif in the cytoplasmic domain of ß1 integrin subunits on endosomes. Silencing Rabgap1 in mouse fibroblasts leads to the intracellular accumulation of active ß1 integrins, alters focal adhesion formation, and decreases cell migration and cancer cell invasion. Functionally, Rabgap1 facilitates active ß1 integrin recycling to the plasma membrane through attenuation of Rab11 activity. Taken together, our results identify Rabgap1 as an important factor for conformation-specific integrin trafficking and define the role of Rabgap1 in ß1-integrin-mediated cell migration in mouse fibroblasts and breast cancer cells.

Breslow thickness 2.0: Why gene expression profiling is a step toward better patient selection for sentinel lymph node biopsies.

Risk-stratification of cutaneous melanoma is important. Patients want to know what to expect after diagnosis, and physicians need to decide on a treatment plan. Historically, melanoma that had spread beyond the skin and regional lymph nodes was largely incurable, and the only approach to preventing a bad outcome was surgery. Through the seminal work of Alexander Breslow and Donald Morton, a system was devised to carefully escalate surgery based on primary tumor thickness and sentinel lymph node status. Today, we know that prophylactic lymph node dissections do not improve survival, but we continue to appreciate the prognostic implications of a positive sentinel node and the benefits of removing nodal metastases, which facilitates locoregional disease control. However, the question arises whether we can better select patients for sentinel lymph node biopsies (SLNB) as, currently, 85% of these procedures are negative and non-therapeutic. Here, we argue that gene expression profiling (GEP) of the diagnostic biopsy is a valuable step toward better patient selection when combined with reliable clinicopathologic (CP) information such as patient age and Breslow thickness. Recently, a CP-GEP-based classifier of nodal metastasis risk, the Merlin Assay, has become commercially available. While CP-GEP is still being validated in prospective studies, preliminary data suggest that it is an independent predictor of nodal metastasis, outperforming clinicopathological variables. The hunt is on for Breslow thickness 2.0.

Differential proteomic expression in indolent vulvar lichen sclerosus, transforming vulvar lichen sclerosus and normal vulvar tissue.

Vulvar lichen sclerosus (VLS) confers approximately 3% risk of malignant transformation to vulvar squamous cell carcinoma (VSCC). We used unbiased proteomic methods to identify differentially expressed proteins in tissue of patients with VLS who developed VSCC compared to those who did not. We used laser capture microdissection- and nanoLC-tandem mass spectrometry to assess protein expression in individuals in normal vulvar tissue (NVT, n = 4), indolent VLS (no VSCC after at least 5 years follow-up, n = 5) or transforming VSCC (preceding VSCC, n = 5). Interferon-γ and antigen-presenting pathways are overexpressed in indolent and transforming VLS compared to NVT. There was differential expression of malignancy-related proteins in transforming VLS compared to indolent VLS (CAV1 overexpression, AKAP12 underexpression), particularly in the EIF2 translation pathway, which has been previously implicated in carcinogenesis. Results of this study provide additional molecular evidence supporting the concept that VLS is a risk factor for VSCC and highlights possible future biomarkers and/or therapeutic targets.

Clinicopathologic models predicting non-sentinel lymph node metastasis in cutaneous melanoma patients: Are they useful for patients with a single positive sentinel node?

BACKGROUND AND OBJECTIVES: Of clinically node-negative (cN0) cutaneous melanoma patients with sentinel lymph node (SLN) metastasis, between 10% and 30% harbor additional metastases in non-sentinel lymph nodes (NSLNs). Approximately 80% of SLN-positive patients have a single positive SLN. METHODS: To assess whether state-of-the-art clinicopathologic models predicting NSLN metastasis had adequate performance, we studied a single-institution cohort of 143 patients with cN0 SLN-positive primary melanoma who underwent subsequent completion lymph node dissection. We used sensitivity (SE) and positive predictive value (PPV) to characterize the ability of the models to identify patients at high risk for NSLN disease. RESULTS: Across Stage III patients, all clinicopathologic models tested had comparable performances. The best performing model identified 52% of NSLN-positive patients (SE = 52%, PPV = 37%). However, for the single SLN-positive subgroup (78% of cohort), none of the models identified high-risk patients (SE > 20%, PPV > 20%) irrespective of the chosen probability threshold used to define the binary risk labels. Thus, we designed a new model to identify high-risk patients with a single positive SLN, which achieved a sensitivity of 49% (PPV = 26%). CONCLUSION: For the largest SLN-positive subgroup, those with a single positive SLN, current model performance is inadequate. New approaches are needed to better estimate nodal disease burden of these patients.

Deep learning for dermatologists: Part II. Current applications.

Because of a convergence of the availability of large data sets, graphics-specific computer hardware, and important theoretical advancements, artificial intelligence has recently contributed to dramatic progress in medicine. One type of artificial intelligence known as deep learning has been particularly impactful for medical image analysis. Deep learning applications have shown promising results in dermatology and other specialties, including radiology, cardiology, and ophthalmology. The modern clinician will benefit from an understanding of the basic features of deep learning to effectively use new applications and to better gauge their utility and limitations. In this second article of a 2-part series, we review the existing and emerging clinical applications of deep learning in dermatology and discuss future opportunities and limitations. Part 1 of this series offered an introduction to the basic concepts of deep learning to facilitate effective communication between clinicians and technical experts.

Deep learning for dermatologists: Part I. Fundamental concepts.

Artificial intelligence is generating substantial interest in the field of medicine. One form of artificial intelligence, deep learning, has led to rapid advances in automated image analysis. In 2017, an algorithm demonstrated the ability to diagnose certain skin cancers from clinical photographs with the accuracy of an expert dermatologist. Subsequently, deep learning has been applied to a range of dermatology applications. Although experts will never be replaced by artificial intelligence, it will certainly affect the specialty of dermatology. In this first article of a 2-part series, the basic concepts of deep learning will be reviewed with the goal of laying the groundwork for effective communication between clinicians and technical colleagues. In part 2 of the series, the clinical applications of deep learning in dermatology will be reviewed and limitations and opportunities will be considered.

Whole-exome sequencing of transforming oral lichen planus reveals mutations in DNA damage repair and apoptosis pathway genes.

BACKGROUND: Oral lichen planus confers a 1% risk of transformation to oral squamous cell carcinoma. While prior exome sequencing studies have identified multiple genetic mutations in oral squamous cell carcinoma, mutational analyses of lichen planus-derived OSCC are lacking. We sought to clarify genomic events associated with oral lichen planus transformation. METHODS: Using rigorous diagnostic criteria, we retrospectively identified patients with non-transforming oral lichen planus (i.e., known to be non-transforming with 5 years of clinical follow-up; n = 17), transforming oral lichen planus (tissue marginal to oral squamous cell carcinoma, n = 9), or oral squamous cell carcinoma arising in lichen planus (n = 17). Gene mutational profiles derived from whole-exome sequencing on fixed mucosal specimens were compared among the groups. RESULTS: The four most frequently mutated genes in transforming oral lichen planus and oral squamous cell carcinoma (TP53, CELSR1, CASP8, and KMT2D) identified 12/17 (71%) of oral squamous cell carcinomas and 5/9 (56%) of transforming oral lichen planus but were absent in non-transforming oral lichen planus. We identified other known oral squamous cell carcinoma mutations (TRRAP, OBSCN, and LRP2) but also previously unreported mutations (TENM3 and ASH1L) in lichen planus-associated oral squamous cell carcinomas. CONCLUSIONS: These findings suggest alterations in DNA damage response and apoptosis pathways underlie lichen planus-related oral squamous cell carcinoma transformation and are supported by mutational signatures indicative of DNA damage. This study characterized patterns of mutational events present in oral lichen planus associated with squamous cell carcinoma and in squamous cell carcinoma associated with oral lichen planus but not in non-transforming oral lichen planus.

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)-kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.

Dermatologic manifestations of solid organ transplantation-associated graft-versus-host disease: A systematic review.

BACKGROUND: Graft-versus-host-disease (GVHD) after solid organ transplantation (SOT) is extremely rare. OBJECTIVE: To investigate the dermatologic manifestations and clinical outcomes of SOT GVHD. METHODS: Systematic literature review of SOT GVHD. RESULTS: After full-text article review, we included 61 articles, representing 115 patients and 126 transplanted organs. The most commonly transplanted organ was the liver (n = 81). Among 115 patients, 101 (87.8%) developed skin involvement. The eruption appeared an average of 48.3 days (range, 3-243 days) posttransplant and was pruritic in 5 of 101 (4.9%) cases. The eruption was described as morbilliform in 2 patients (1.9%), confluent in 6 (5.9%), and desquamative in 4 (3.9%) cases. In many cases, specific dermatologic descriptions were lacking. The mortality rate was 72.2%. Relative time of death was reported in 23 patients who died during the follow-up period. These patients died an average of 99.2 days (range, 22-270 days) posttransplant, or 50.9 days after the appearance of dermatologic symptoms. Frequent causes of death were sepsis and multiorgan failure. LIMITATIONS: Incomplete descriptions of skin findings and potential publication bias resulting in publication of only the most severe cases. CONCLUSIONS: GVHD is a potentially fatal condition that can occur after SOT and often presents with a skin rash. We recommend that dermatologists have a low threshold to consider and pursue this diagnosis in the setting of post-SOT skin eruption.

Clinical and histopathologic manifestations of solid organ transplantation-associated graft-versus-host disease involving the skin: A single-center retrospective study.

BACKGROUND: Graft-versus-host disease (GVHD) following solid organ transplantation (SOT) is extremely rare and infrequently described in the dermatologic literature. METHODS: We performed a retrospective clinicopathologic review of our institution's experience with patients diagnosed with SOT-associated GVHD (SOT GVHD) (May 1, 1996 to September 1, 2017). RESULTS: Of nine patients with SOT GVHD, seven had undergone liver transplantation, while two had undergone lung transplantation. All presented initially with a skin eruption, which developed an average of 63 days (range: 11-162 days) post transplant. The average time to diagnosis following the onset of the skin eruption was 12 days (range: 0-54 days). Diagnosis was often delayed because of a competing diagnosis of drug reaction. Frequent skin findings included pruritic erythematous to violaceous macules and papules with desquamation. Histopathology showed vacuolar interface dermatitis in 12 of 15 cases (80.0%). Of the 11 specimens in which a hair follicle was present for evaluation, vacuolar interface changes around the hair follicle were present in eight (72.7%) cases. Seven patients (77.8%) died from complications during the follow-up period. CONCLUSIONS: SOT GVHD presents initially with skin involvement, is associated with vacuolar interface changes on skin biopsy, and is associated with a high mortality rate. Clinicopathologic correlation is required for accurate diagnosis.

Hepatic adenomas with synchronous or metachronous fibrolamellar carcinomas: both are characterized by LFABP loss.

Rare hepatic adenomas are associated with synchronous or metachronous fibrolamellar carcinomas. The morphology of these adenomas has not been well described and they have not been subclassifed using the current molecular classification schema. We examined four hepatic adenomas co-occurring with or preceding a diagnosis of fibrolamellar carcinoma in three patients. On histological examination, three of the adenomas showed the typical morphology of HNF1-α inactivated adenomas, whereas one showed a myxoid adenoma morphology. All of the adenomas were negative for PRKACA rearrangements by Fluorescence in situ Hybridization (FISH) analysis. All four of the adenomas showed complete loss or significant reduction of liver fatty acid binding protein (LFABP) expression by immunohistochemistry. Interestingly, the fibrolamellar carcinomas in each case also showed loss of LFABP by immunohistochemistry. One of the fibrolamellar carcinomas was negative for PRKACA rearrangements by FISH, whereas the others were positive. To investigate if LFBAP loss is typical of fibrolamellar carcinomas in general, an additional cohort of tumors was studied (n=19). All 19 fibrolamellar carcinomas showed the expected PRKACA rearrangements and immunostains showed loss of LFABP in each case, consistent with HNF1-α inactivation. To validate this observation, mass spectrometry-based proteomics was performed on tumor-normal pairs of six fibrolamellar carcinomas and showed an average 10-fold reduction in LFABP protein levels, compared with matched normal liver tissue. In conclusion, hepatic adenomas co-occurring with fibrolamellar carcinomas show LFABP loss and are negative for PRKACA rearrangements, indicating they are genetically distinct lesions. These data also demonstrate that LFABP loss, which characterizes HNF1-α inactivation, is a consistent feature of fibrolamellar carcinoma, indicating HNF1-α inactivation is an important event in fibrolamellar carcinoma pathogenesis.

Beta1 integrin cytoplasmic tyrosines promote skin tumorigenesis independent of their phosphorylation.

ß1 integrin tyrosine phosphorylation by oncogenic kinases, such as Src, has been predicted to induce tumorigenesis by disrupting adhesion and modifying integrin signaling. We directly tested this hypothesis by subjecting mice with "nonphosphorylatable" tyrosine-to-phenylalanine substitutions in the conserved ß1 cytoplasmic tail NPxY motifs to a model of cutaneous carcinogenesis in the presence or absence of elevated Src activity. We found that hydrophobic phenylalanine substitutions of both tyrosines diminished the binding of tail-interacting proteins, including talins and kindlins, resulting in reduced ß1-mediated adhesion, focal adhesion kinase (FAK) signaling, and epidermal progenitor cell-derived skin tumors. However, increased Src activity drove tumor formation independent of the phenylalanine substitutions by enhancing FAK activity, which in turn maintained the epidermal progenitor state and blocked keratinocyte differentiation. We conclude that a Src/FAK signaling unit inhibits differentiation to promote tumorigenesis downstream of ß1 integrin and independent of ß1 integrin tyrosine phosphorylation.

Minimally invasive surgical approach to model hypertrophic scarring in the rabbit ear.

BACKGROUND: Current strategies for hypertrophic scar prevention and treatment are limited. OBJECTIVE: To facilitate these efforts, a minimally invasive hypertrophic scar model was created in a rabbit ear for the first time based on previous methods used to induce ischemia. METHODS: Six New Zealand white rabbits (12 ears total) were studied. First, ischemia was achieved by ligating the cranial artery, cranial vein and central artery, while preserving the caudal artery, caudal vein and central vein, respectively. The relative level of ischemia induced at time of surgery, both baseline and maximum perfusion, was assessed with a fluorescent light-assisted angiography and demonstrated lower rates of perfusion in the ischemic ears. Following vascular injury, a 2-cm full thickness linear wound was created on the ventral ear and closed with 4 - 0 Nylon sutures under high tension. For each rabbit, one ear received a combination of ischemia and wounding with suture tension (n = 6), while the other ear was non-ischemic with wounding and suture tension alone (n = 6). RESULTS: Four weeks post-operatively, ischemic ears developed scar hypertrophy (histological scar thickness: 1.1 ± 0.2 mm versus 0.5 ± 0.1 mm, p < 0.05). CONCLUSION: Herein, we describe a novel, prototypical minimally invasive rabbit ear model of hypertrophic scar formation that can allow investigation of new drugs for scar prevention.

Clinicopathological and cellular senescence biomarkers in chronic stalled wounds.

BACKGROUND: Chronic wounds have been associated with an elevated burden of cellular senescence, a state of essentially irreversible cell cycle arrest, resistance to apoptosis, and a secretory phenotype. However, whether senescent cells contribute to wound chronicity in humans remains unclear. The objective of this article is to assess the role of clinicopathological characteristics and cellular senescence in the time-to-healing of chronic wounds. METHODS: A cohort of 79 patients with chronic wounds was evaluated in a single-center academic practice from February 1, 2005, to February 28, 2015, and followed for up to 36 months. Clinical characteristics and wound biopsies were obtained at baseline, and time-to-healing was assessed. Wound biopsies were analyzed histologically for pathological characteristics and molecularly for markers of cellular senescence. In addition, biopsy slides were stained for p16INK4a expression. RESULTS: No clinical or pathological characteristics were found to have significant associations with time-to-healing. A Cox proportional hazard ratio model revealed increased CDKN1A (p21CIP1/WAF1 ) expression to predict longer time-to-healing, and a model adjusted for gender and epidermal hyperplasia revealed increased CDKN1A expression and decreased PAPPA expression to predict longer time-to-healing. Increased p16INK4a staining was observed in diabetic wounds compared to non-diabetic wounds, and the same association was observed in the context of high dermal fibrosis. CONCLUSIONS: The findings of this pilot study suggest that senescent cells contribute to wound chronicity in humans, especially in diabetic wounds.

Pentamidine-loaded gelatin decreases adhesion formation of flexor tendon.

Background: Prevention of adhesion formation following flexor tendon repair is essential for restoration of normal finger function. Although many medications have been studied in the experimental setting to prevent adhesions, clinical application is limited due to the complexity of application and delivery in clinical translation. Methods: In this study, optimal dosages of gelatin and pentamidine were validated by gelatin concentration test. Following cell viability, cell migration, live and dead cell, and cell adhesion assay of the Turkey tenocytes, a model of Turkey tendon repair was established to evaluate the effectiveness of the Pentamidine-Gelatin sheet. Results: Pentamidine carried with gelatin, a Food and drug administration (FDA) approved material for drug delivery, showed good dynamic release, biocompatibility, and degradation. The optimal dose of pentamidine (25ug) was determined in the in vivo study using tenocyte viability, migration, and cell adhesion assays. Further biochemical analyses demonstrated that this positive effect may be due to pentamidine downregulating the Wnt signaling pathway without affecting collagen expression. Conclusions: We tested a FDA-approved antibiotic, pentamidine, for reducing adhesion formation after flexor tendon repair in both in vitro and in vivo using a novel turkey animal model. Compared with the non-pentamidine treatment group, pentamidine treated turkeys had significantly reduced adhesions and improved digit function after six weeks of tendon healing. The translational potential of this article: This study for the first time showed that a common clinical drug, pentamidine, has a potential for clinical application to reduce tendon adhesions and improve tendon gliding function without interfering with tendon healing.

CP-GEP (Merlin) gene expression profiling: can my melanoma patient forgo sentinel lymph node biopsy?

Melanoma risk stratification is crucial for both patients and physicians. Patients want to understand what to expect after diagnosis, and physicians need to decide on an appropriate treatment plan. Traditionally, risk stratification has been based on Breslow thickness and sentinel lymph node (SLN) status. However, the introduction of CP-GEP (Merlin) has provided a molecular test that can be performed on primary melanoma diagnostic biopsy tissue, offering additional risk stratification beyond established variables. In this review, we assess the utility of CP-GEP testing compared to clinicopathologic variables and SLN status and propose a multilayered approach to selecting patients for adjuvant therapy using CP-GEP technology.