A Novel Multi-Component Formulation Reduces Inflammation In Vitro and Clinically Lessens the Symptoms of Chronic Eczematous Skin.

Long-term treatments for inflammatory skin diseases like atopic dermatitis or eczema can cause adverse effects. Super Protein Multifunction (SPM) was investigated as a potential treatment for managing skin inflammation by monitoring the expression of pro-inflammatory cytokines induced using LPS and poly(I:C)/TNFα in HaCaT keratinocytes and Hs27 fibroblasts as measured via RT-PCR. SPM solution was also assessed for its effect on cytokine release, measured using ELISA, in a UVB-irradiated 3D human skin model. To evaluate the efficiency of SPM, 20 patients with mild eczematous skin were randomized to receive SPM or vehicle twice a day for three weeks in a double-blind controlled trial. In vitro studies showed SPM inhibited inflammation-induced IL-1ß, IL-6, IL-33, IL-1α, TSLP, and TNFα expression or release. In the clinical study, the SPM group showed significant improvements in the IGA, PA, and DLQI scores compared to the vehicle group. Neither group showed significant differences in VAS (pruritus). Histological analysis showed reduced stratum corneum thickness and inflammatory cell infiltration. The results suggest that SPM may reduce inflammation in individuals with chronic eczematous skin.

Inhibition of Melanoma Cell-Intrinsic Tim-3 Stimulates MAPK-Dependent Tumorigenesis.

T-cell immunoglobulin mucin family member 3 (Tim-3) is an immune checkpoint receptor that dampens effector functions and causes terminal exhaustion of cytotoxic T cells. Tim-3 inhibitors are under investigation in immuno-oncology (IO) trials, because blockade of T-cell-Tim-3 enhances antitumor immunity. Here, we identify an additional role for Tim-3 as a growth-suppressive receptor intrinsic to melanoma cells. Inhibition of melanoma cell-Tim-3 promoted tumor growth in both immunocompetent and immunocompromised mice, while melanoma-specific Tim-3 overexpression attenuated tumorigenesis. Ab-mediated Tim-3 blockade inhibited growth of immunogenic murine melanomas in T-cell-competent hosts, consistent with established antitumor effects of T-cell-Tim-3 inhibition. In contrast, Tim-3 Ab administration stimulated tumorigenesis of both highly and lesser immunogenic murine and human melanomas in T-cell-deficient mice, confirming growth-promoting effects of melanoma-Tim-3 antagonism. Melanoma-Tim-3 activation suppressed, while its blockade enhanced, phosphorylation of pro-proliferative downstream MAPK signaling mediators. Finally, pharmacologic MAPK inhibition reversed unwanted Tim-3 Ab-mediated tumorigenesis in T-cell-deficient mice and enhanced desired antitumor activity of Tim-3 interference in T-cell-competent hosts. These results identify melanoma-Tim-3 blockade as a mechanism that antagonizes T-cell-Tim-3-directed IO therapeutic efficacy. They further reveal MAPK targeting as a combination strategy for circumventing adverse consequences of unintended melanoma-Tim-3 inhibition. SIGNIFICANCE: Tim-3 is a growth-suppressive receptor intrinsic to melanoma cells, the blockade of which promotes MAPK-dependent tumorigenesis and thus counteracts antitumor activity of T-cell-directed Tim-3 inhibition.

Topical therapy for regression and melanoma prevention of congenital giant nevi.

Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.

Stepwise-edited, human melanoma models reveal mutations' effect on tumor and microenvironment.

Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.

Impact of Next-generation Sequencing on Interobserver Agreement and Diagnosis of Spitzoid Neoplasms.

Atypical Spitzoid melanocytic tumors are diagnostically challenging. Many studies have suggested various genomic markers to improve classification and prognostication. We aimed to assess whether next-generation sequencing studies using the Tempus xO assay assessing mutations in 1711 cancer-related genes and performing whole transcriptome mRNA sequencing for structural alterations could improve diagnostic agreement and accuracy in assessing neoplasms with Spitzoid histologic features. Twenty expert pathologists were asked to review 70 consultation level cases with Spitzoid features, once with limited clinical information and again with additional genomic information. There was an improvement in overall agreement with additional genomic information. Most significantly, there was increase in agreement of the diagnosis of conventional melanoma from moderate (κ=0.470, SE=0.0105) to substantial (κ=0.645, SE=0.0143) as measured by an average Cohen κ. Clinical follow-up was available in all 70 cases which substantiated that the improved agreement was clinically significant. Among 3 patients with distant metastatic disease, there was a highly significant increase in diagnostic recognition of the cases as conventional melanoma with genomics (P<0.005). In one case, none of 20 pathologists recognized a tumor with BRAF and TERT promoter mutations associated with fatal outcome as a conventional melanoma when only limited clinical information was provided, whereas 60% of pathologists correctly diagnosed this case when genomic information was also available. There was also a significant improvement in agreement of which lesions should be classified in the Spitz category/WHO Pathway from an average Cohen κ of 0.360 (SE=0.00921) to 0.607 (SE=0.0232) with genomics.

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.

Peripheral host T cells survive hematopoietic stem cell transplantation and promote graft-versus-host disease.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67+) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

Photochemical Tissue Passivation Prevents Contracture of Full Thickness Wounds in Mice.

BACKGROUND AND OBJECTIVES: Wound contracture formation from excessive myofibroblast activity can result in debilitating morbidities. There are currently no treatments to prevent contracture. Photochemical tissue passivation (PTP), an established, safe, and user-friendly treatment modality, crosslinks collagen by a light-activated process, thus modulating the wound healing response and scarring. We hypothesised that PTP treatment would reinforce wounds by blunting the fibrotic response thus limiting contracture. STUDY DESIGN/MATERIALS AND METHODS: Full-thickness, 1 cm × 1 cm excisional wounds were created on the dorsum of 32 C57BL/6 mice. Treated wounds were painted with photosensitizing dye and exposed to visible light. Wounds were serially photographed over 6 weeks to measure wound contracture. At 7, 14, 21, and 42 days after wound creation, mice were euthanized and wounds were harvested for histologic review by a dermatopathologist. RESULTS: By Day 7, control wounds had significantly more contracture than those treated with PTP (33.0 ± 17.1% and 19.3 ± 9.0%, respectively; P = 0.011). PTP-treated wounds maintained approximately 20% less contracture than controls from Day 14 and on (P < 0.05). By Day 42, wounds had contracted by 86.9 ± 5.5% in controls and 64.2 ± 3.2% in PTP-treated wounds (P < 0.03). Histologically, PTP wounds had earlier growth and development of dermal collagen, neovascularization, and development of skin appendages, compared with control wounds. CONCLUSIONS: PTP significantly limits contracture of full-thickness wounds and improves wound healing. PTP-treated wounds histologically demonstrate more mature structural organization than untreated wounds and closely resemble native skin. PTP treatment may be applicable not only for excisional wounds, but also for wounds with a high incidence of contracture and associated morbidity. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

The Pathogenesis of Port Wine Stain and Sturge Weber Syndrome: Complex Interactions between Genetic Alterations and Aberrant MAPK and PI3K Activation.

Port wine stain (PWS) is a congenital vascular malformation involving human skin. Approximately 15-20% of children a facial PWS involving the ophthalmic (V1) trigeminal dermatome are at risk for Sturge Weber syndrome (SWS), a neurocutaneous disorder with vascular malformations in the cerebral cortex on the same side of the facial PWS lesions. Recently, evidence has surfaced that advanced our understanding of the pathogenesis of PWS/SWS, including discoveries of somatic genetic mutations (GNAQ, PI3K), MAPK and PI3K aberrant activations, and molecular phenotypes of PWS endothelial cells. In this review, we summarize current knowledge on the etiology and pathology of PWS/SWS based on evidence that the activation of MAPK and/or PI3K contributes to the malformations, as well as potential futuristic treatment approaches targeting these aberrantly dysregulated signaling pathways. Current data support that: (1) PWS is a multifactorial malformation involving the entire physiological structure of human skin; (2) PWS should be pathoanatomically re-defined as "a malformation resulting from differentiation-impaired endothelial cells with a progressive dilatation of immature venule-like vasculatures"; (3) dysregulation of vascular MAPK and/or PI3K signaling during human embryonic development plays a part in the pathogenesis and progression of PWS/SWS; and (4) sporadic low frequency somatic mutations, such as GNAQ, PI3K, work as team players but not as a lone wolf, contributing to the development of vascular phenotypes. We also address many crucial questions yet to be answered in the future research investigations.

Atypical Cellular Blue Nevus of the Foot: A Case Report.

Blue nevus is a congenital and acquired melanocytic proliferation that includes different histological types. The atypical cellular type has been rarely described and it classically has a benign course. However, because of its intermediate features between common blue nevus and malignant blue nevus, long-term clinical follow-up is required. Here we report the case of a 28-year-old woman who presented with an atypical cellular blue nevus on the right foot.

Continuous Spatial Sequences of Lichen Sclerosus, Penile Intraepithelial Neoplasia, and Invasive Carcinomas: A Study of 109 Cases.

Lichen sclerosus (LSc) with penile cancer is found in about two thirds of specimens. It has been hypothesized that LSc represents a precancerous condition. To qualify as such, in addition to cytological atypia and similarity with the invasive tumor, a spatial correlation between LSc and neoplastic lesions needs to be demonstrated. The purpose of this study was to evaluate such a spatial relationship. Circumcision (28 cases) and penectomy (81 cases) specimens were evaluated. All cases had LSc, penile intraepithelial neoplasia (PeIN), and/or invasive squamous cell carcinomas. We examined LSc in relation to invasive carcinoma, PeIN, and normal epithelia. Invasive squamous cell carcinomas, classified according to the World Health Organization criteria as non-human papillomavirus (HPV)-related and HPV-related PeIN, were present in 100 cases. Non-HPV-related (differentiated) PeIN was the most common subtype associated with LSc (89%). There were 5 spatial patterns identified: (1) LSc adjacent to PeIN (23%), (2) LSc adjacent and comprising PeIN (42%), (3) LSc next to and within invasive carcinomas (8%), (4) LSc throughout the sequence PeIN-invasive carcinoma (24%), and (5) LSc was separate (with normal tissue between the lesions) from PeIN and/or invasive carcinomas in a minority of cases (3%). LSc within the cancer was not previously described. In this series, we found 35 cases with LSc within invasive carcinomas. The striking continuous spatial relationship among LSc, PeIN, and/or invasive carcinoma as shown in this study may be a necessary (but not sufficient) condition for the hypothesis postulating LSc as a penile precancerous lesion.

Membrane trafficking and exocytosis are upregulated in port wine stain blood vessels.

INTRODUCTION: Port wine stain (PWS) is characterized as a progressive dilatation of immature venule-like vasculatures which result from differentiation-impaired endothelial cells. In this study, we aimed to identify the major biological pathways accounting for the pathogenesis of PWS. METHODS: Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to identify differentially expressed proteins in PWS lesions, followed by confirmative studies with immunohistochemistry, immunoblot and transmission electron microscopy (TEM). RESULTS: 107 out of 299 identified proteins showed differential expressions in PWS lesions as compared to normal skin, mainly involving the functions of biosynthesis, membrane trafficking, cytoskeleton and cell adhesion/migration. The confirmative studies showed that expressions of membrane trafficking/exocytosis related proteins such as VAT1, IQGAP1, HSC70, clathrin, perlecan, spectrin α1 and GDIR1 were significantly increased in PWS blood vessels as compared to normal ones; while collagen subtypes 6A1 and 6A3 were decreased in PWS skin. Furthermore, TEM studies showed there is a significant upregulation of extracellular vesicle exocytosis from PWS blood vessels as compared to control. CONCLUSIONS: The biological process of membrane trafficking and exocytosis is enhanced in PWS blood vessels. Our results imply that the extracellular vesicles released by lesional endothelial cells may act as potential intercellular signaling mediators to contribute to the pathogenesis of PWS.

Histological features and outcome of inverted type-A melanocytic nevi.

The presence of enlarged epithelioid/spindled nests located deep in the reticular dermis of a biphasic melanocytic neoplasm can mimic melanoma arising in a pre-existing nevus, causing over-interpretation of malignancy. We aimed to define the clinicopathologic significance of epithelioid/spindled nests in melanocytic nevi. Retrospectively using clinical and histologic information, we characterized 121 patients with a single lesion showing epithelioid/spindled melanocytes in the reticular dermis or subcutaneous fat, surrounded by melanophages, sometimes blending in with the adnexa. The majority of nevi occurred in women in the ages of 10 to 39 years, where the most frequent presentation was a changing mole. While 78% of the lesions displayed an anatomic (Clark's) level of IV-V, there was no ulceration, significant regression or inflammation. Up to 2 mitoses were found in only 12% of the cases, not correlating with the severity of cytological atypia. No recurrence or metastasis occurred during 45.5 months (mean) of clinical follow up in 26 patients. Notwithstanding the deep dermal extension, these findings suggest a benign histopathology and clinical outcome. Having compared the overlapping histopathology and clinical features between deep penetrating/clonal nevus and combined nevus, we posit that "inverted type-A nevus" might be considered a variant of the two.

Skin regeneration with all accessory organs following ablation with irreversible electroporation.

Skin scar formation is a complex process that results in the formation of dense extracellular matrix (ECM) without normal skin appendages such as hair and glands. The absence of a scarless healing model in adult mammals prevents the development of successful therapies. We show that irreversible electroporation of skin drives its regeneration with all accessory organs in normal adult rats. Pulsed electric fields at 500 V, with 70 µs pulse duration and 1000 pulses delivered at 3 Hz, applied through two electrodes separated by 2 mm lead to massive cell death. However, the ECM architecture of the skin was preserved. Six months after the ablation, the epidermis, sebaceous glands, panniculus carnosus, hair follicles, microvasculature and arrector pili muscle were altogether re-formed in the entire ablated area. These results suggest a key role of the ECM architecture in the differentiation, migration and signalling of cells during scarless wound healing. Copyright © 2016 John Wiley & Sons, Ltd.

New insights into naevoid melanomas: a clinicopathological reassessment.

AIMS: Because the term 'naevoid melanoma' has variable clinical and pathological interpretations, we aimed to clarify the features of melanomas referred to as naevoid. METHODS AND RESULTS: A review was undertaken of 102 melanomas diagnosed histopathologically as naevoid melanomas and ascertained by European Organization for Research and Treatment of Cancer Melanoma Group Subcommittee pathologists from their records. We found these could be classified morphologically into three groups. Thirteen melanomas were overlying genuine naevi and were therefore excluded. Of the 89 melanomas considered to be naevoid, 11 presented clinically as exophytic papillomatous nodules with little junctional component and composed of small atypical cells showing numerous mitoses and no change with depth; we termed these 'papillomatous naevoid' melanomas. The other 78 were flat or only slightly raised, and had a superficial spreading melanoma-like component with maturation to a small cell, but still an atypical, dermal component; we termed these 'maturing naevoid' melanomas. We showed that papillomatous and maturing naevoid melanomas also have differing immunochemical profiles. Preliminary clinical follow-up suggested different outcomes for these two naevoid melanoma types. CONCLUSIONS: Melanomas that have been classified as naevoid melanomas comprise two types with distinct clinical, histopathological and immunohistochemical features that may also be prognostically significant.

The correlation of the standard 5 probe FISH assay with melanocytic tumors of uncertain malignant potential.

BACKGROUND: FISH has recently emerged as a technique to better assess the malignant potential of histologically ambiguous melanocytic lesions. However, the usefulness of FISH has not been conclusively established. The purpose of this study was to further explore the diagnostic value of FISH in distinguishing the borderline melanocytic tumor (BMT) from melanoma. METHOD: 73 cases with BMT were analyzed retrospectively from a dermatopathology database between 2010-2015. FISH studies were conducted in each case using probes targeting 5 loci including CCND1 on 11q13, RREB1 on 6p25, MYB on 6q23, CDKN2A on 9p21, and CEP 6 control probe for chromosome 6. RESULTS: The study was composed of 50 females and 23 males with an age range of 1-73 and a mean age of 35years. Of the 6 cases in the superficial atypical Spitz tumor (AST) category, 2 had indeterminate results due to polyploidy. In the conventional atypical Spitz tumor cases, FISH was positive in 3 of 15 cases. Of the 27 cases in the borderline nevoid tumor (BNM) category, 3 showed positive FISH and 3 were equivocal due to the possibility of polyploidy. 3 of 13 cases of the borderline tumor of deep penetrating nevus variant (B-DPN) were positive for FISH. Neither of the 2 pigmented epithelioid melanocytoma (PEM) cases had positive FISH result. Of the 4 cases in the superficial atypical dermoepidermal nevomelanocytic proliferation group, only 1 met the FISH diagnostic criteria for melanoma. None of the 6 borderline tumors with overlapping features met FISH criteria diagnostic of melanoma. Clinical follow up was available on 55 patients. None of the patients had recurrence nor died of the disease. Lymph node biopsy was performed on five patients without evidence of metastasis. CONCLUSION: Despite the benefits of FISH, it is limited by the fact that melanomas are not genetically identical whereby certain genetic abnormalities are only seen in specific subtypes. Additionally, FISH only targets specific chromosomes resulting in limitations in sensitivity and specificity. Although FISH has proven to be highly sensitive and specific in distinguishing unequivocally benign from malignant lesions, in cases of histopathological ambiguity, these parameters cannot be assessed with great confidence because the histopathological diagnosis (gold standard) is not without uncertainty. The 4-probe set (excluding 9p21) consistently showed chromosomal aberrations throughout all groups, but only 10 of the 73 total cases (13%) met the diagnostic criteria for melanoma. Moreover, it would be wise to establish new cytogenetic reference values that incorporate these borderline lesions in an effort to better assess the possibility of malignant behavior and or define a cytogenetic profile supportive of its categorization as an indeterminate proliferation. Polyploidy is another inherent limitation, which leads to false positives due to the absolute signal counts incorrectly reflecting relative imbalances in the tumor genome.

A 12-Month-Old Healthy Girl with a New Oral Ulcer and Chronic Diaper Rash.

A 12-month-old healthy girl presented with a chronic diaper rash. Physical examination demonstrated crusting of the scalp, erythematous papules with surrounding petechiae on the lower abdomen, and an intraoral palatal ulcer. Further imaging demonstrated bone involvement. Histopathologic examination of involved skin and the intraoral ulcer demonstrated epithelioid histiocytes with "coffee bean-shaped" nuclei, staining positive for CD1a and langerin by immunohistochemistry, consistent with Langerhans cell histiocytosis (LCH). LCH is a disease entity of unknown etiology characterized by histiocytic proliferation that most commonly presents in young children. The cutaneous findings of LCH include a seborrheic dermatitis-like and/or red-brown papular eruption. Intraoral examination is crucial as oral mucosal and maxillofacial skeletal disease can also be seen in LCH. When a child presents with a recalcitrant seborrheic dermatitis-like eruption or chronic diaper rash, the clinician should be alerted to the possibility of LCH. Timely recognition and diagnosis of LCH is important for oncologic referral, evaluation, and treatment.