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Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.

Ulceration of melanoma is associated with neutrophil infiltrates and lower survival rates opposite to non-ulcerated melanoma. Neutrophils release neutrophil extracellular traps (NETs) that are chromatin structures loaded with antimicrobial proteins. Since NETs have been correlated with tumor progression, we investigated whether NETs appear in melanoma and affect melanoma cells. Indeed, human primary melanoma biopsies revealed neutrophils releasing NETs in all of 27 ulcerated melanomas, whereas NETs were absent in all of 7 non-ulcerated melanomas. However, the quantity of intratumoral NETs did not correlate with tumor progression of melanoma. Interestingly, in vitro assays showed that melanoma cells attach to NETs via integrin-mediated adhesion and that NETs inhibit tumor cell migration. Moreover, co-culturing of NETs and melanoma cells had a cytotoxic effect on melanoma cells resulting in necrosis. Hence, we discovered in vitro an antineoplastic role of NETs in melanoma., (© 2019 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Background: In 2009, the AJCC issued a revised melanoma staging system. In addition to tumor thickness and ulceration, the mitotic rate was introduced as the third major prognostic parameter for the classification of primary cutaneous melanoma. Given that, according to the 2009 AJCC classification, the detection of one or more dermal tumor mitoses leads to an upstaging - from stage Ia to Ib - of melanomas with a tumor thickness of ≤ 1.0 mm, we set out to investigate the reproducibility of this new parameter., Methods: In order to assess interobserver reliability, 17 dermatopathologists und pathologists - all well versed in the diagnosis of cutaneous melanoma - analyzed the mitotic rate in 15 thin primary cutaneous melanomas (mean tumor thickness 0.91 mm) using identical slides. Mitotic rates were determined on H&E and phosphohistone H3 (Ser10)-stained samples. Without knowledge of their previous assessment, five of the aforementioned examiners reevaluated the samples after more than one year in order to ascertain intraobserver reliability., Results: Interobserver reliability of the mitotic rate in thin primary melanomas is disappointing and independent of whether H&E or immunohistochemically stained samples are used (kappa value: 0.088 [H&E], 0.154 [IH], respectively). Kappa values improved to 0.345 (H&E) and 0.403 (IH) when using a cutoff of 0/1 vs. 2+ mitoses. Similarly unsatisfactory, kappa values for intraobserver reliability ranged from 0.18 and 0.348, depending on the individual examiner., Discussion: Given the unsatisfactory reproducibility and large variations in assessing the mitotic rate, it remains a matter of debate whether this diagnostic parameter should play a role in therapeutic decisions., (© 2016 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.)

Prohormone convertases (PCs) are endoproteases that process many substrates in addition to hormone precursors. Although overexpression of PCs is linked to carcinogenesis in some solid tumors, the role of subtilisin-kexin isoenzyme-1 (SKI-1) in this context is unknown. We show that SKI-1 is constitutively expressed in human pigment cells with higher SKI activity in seven out of eight melanoma cell lines compared with normal melanocytes. SKI-1 immunoreactivity is also detectable in tumor cells of melanoma metastases. Moreover, tissue samples of the latter display higher SKI-1 mRNA levels and activity than normal skin. From various stimuli tested, 12-O-tetradecanoylphorbol-13-acetate and tunicamycin affected SKI-1 expression. Importantly, SKI-1 inhibition by the cell-permeable enzyme inhibitor decanoyl-RRLL-chloromethylketone (dec-RRLL-CMK) not only suppressed proliferation and metabolic activity of melanoma cells in vitro but also reduced tumor growth of melanoma cells injected intracutaneously into immunodeficient mice. Mechanistic studies revealed that dec-RRLL-CMK induces classical apoptosis of melanoma cells in vitro and affects expression of several SKI-1 target genes including activating transcription factor 6 (ATF6). However, ATF6 gene silencing does not result in apoptosis of melanoma cells, suggesting that dec-RRLL-CMK induces cell death in an ATF6-independent manner. Our findings encourage further studies on SKI-1 as a potential target for melanoma therapy.

Background: Uveal melanomas represent 3.1% of all melanomas, with a high potential of metastatic disease of up to 50%, where the median survival time is 6 months. Though liver metastases dominate as the primary site for metastasis, the existence of primary skin metastases is still under discussion but has been reported in only a few studies., Objectives: We present two cases in which patients with a known history of uveal melanoma developed melanoma skin metastases., Methods: Mutational analysis was performed to clarify the origin of the metastases (uvea or skin)., Results: The analyses revealed GNA11 mutations, which are typical for uveal melanoma. These cases strongly suggest the skin to be the primary site of uveal melanoma., Conclusions: Knowledge about the mutational status of uveal melanomas opens the opportunity for future targeted therapies that directly interact with the mutation and its activated signal cascades. First trials in uveal melanoma have shown promising results with MEK inhibitors., (© 2013 The Authors BJD © 2013 British Association of Dermatologists.)

Background: TNM classifications are the basis for diagnostic and therapeutic procedures in oncology. Histopathological reports have to enable a proper indexing of tumor specific findings into recent classifications., Methods: A systematic review of the literature was performed to identify reports dealing with the assessment of mitotic rate and the processing and evaluation of sentinel node biopsies in malignant melanoma. On the basis of this review an expert panel of dermatopathologists and general pathologists discussed and agreed recommendations for general practice., Results: Following recommendations were agreed with a broad consensus (93-100 % agreement): The determination of the mitotic rate in primary melanoma is performed on HE slides. The evaluation of an area of 1 mm(2) is sufficient. Only dermal mitoses are considered. The counted number of mitoses is provided as an integer value. The mitotic rate shall be determined in primary melanomas of ≤1.00 mm vertical tumor thickness according to the hot-spot method and provided as an integer value in relation to an area of 1 mm(2) . The determination of the mitotic rate in the case of thicker primary melanomas is desirable. In general, for the evaluation of each sentinel lymph node, 4 slides should be prepared. For diagnostic purposes, immunohistochemistry (preferably with antibodies against S100ß, Melan A and HMB-45) should be performed in addition to HE staining. The pathology report should provide information about micro-metastases and their longest extension (one-tenth of a millimeter)., Conclusions: These recommendations are suitable for standardizing the histopathological diagnosis of malignant melanoma and for providing a common basis for clinical decisions and scientific research., (© The Authors • Journal compilation © Blackwell Verlag GmbH, Berlin.)

Melanoma accounts for ∼ 79% of skin cancer-related deaths, and the receptor activator of NF-κB (RANK)-receptor activator of NF-κB ligand (RANKL) pathway has been shown to be involved in the migration and metastasis of epithelial tumor cells. In this study, we demonstrate that RANK was significantly increased in peripheral circulating melanoma cells, primary melanomas, and metastases from stage IV melanoma patients compared with tumor cells from stage I melanoma patients. However, upregulated RANK expression was not found in stage IV melanoma patients with bone metastases compared with stage IV melanoma patients without bone metastases, providing a possible explanation for the clinical observation that melanoma cells do not preferentially metastasize to bone tissue. Strikingly, RANK-expressing melanoma cells from peripheral blood, primary tumors, or metastases of stage IV patients coexpressed ATP-binding cassette (ABC) B5 and CD133, both markers characteristic of melanoma-initiating cells, suggesting a tumor stem cell-like phenotype. In support of this hypothesis, RANK-expressing melanoma cells showed a reduced Ki67 proliferation index compared with RANK(-) melanoma cells from the same patient and are able to induce tumor growth in immunodeficient mice. Together, our data demonstrate that RANK expression is increased in metastatic melanoma and highly upregulated on melanoma-initiating cells, suggesting that RANK might be involved in the development and maintenance of melanoma-initiating cells and possibly in metastatic spreading.

The growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, whereas late stage melanoma cells have often been reported to be "multi-cytokine-resistant." Here, we analyzed the melanoma cell line 1286, resistant towards the growth-inhibitory effects of interleukin 6 (IL-6), and oncostatin M (OSM), to better understand the mechanisms underlying cytokine resistance. Although the relevant receptors gp130 and OSMR are expressed at the cell surface of these cells, cytokine stimulation hardly led to the activation of Janus kinase 1 and signal transducer and activator of transcription (STAT)3 and STAT1. We found a high-level constitutive expression of suppressors of cytokine signaling 3 (SOCS3) that did not further increase after cytokine treatment. Importantly, upon suppression of SOCS3 by short interfering RNA, cells became susceptible towards OSM and IL-6: they showed an enhanced STAT3 phosphorylation and a dramatically increased STAT1 phosphorylation. Moreover, suppression of SOCS3 rendered 1286 cells sensitive to the antiproliferative action of IL-6 and OSM, but not of IFN-alpha. Interestingly, SOCS3-short interfering RNA treatment also increased the growth-inhibitory effect in cytokine-sensitive WM239 cells expressing SOCS3 in an inducible way. Thus, SOCS3 expression confers a growth advantage to these cell lines. Constitutive SOCS3 mRNA expression, although at lower levels than in 1286 cells, was found in nine additional human melanoma cell lines and in normal human melanocytes, although at the protein level, SOCS3 expression was marginal at best. However, in situ analysis of human melanoma specimens revealed SOCS3 immunoreactivity in 3 out of 10 samples, suggesting that in vivo SOCS3 may possibly play a role in IL-6 resistance in at least a fraction of tumors.

Surgery in malignant melanoma of the external ear often leads to substantial defects where reconstruction poses a difficult challenge. We describe an option of a one-step ear reconstruction after subtotal ear resection in malignant melanoma surgery. In a patient with a high-risk melanoma of the helical rim, a wide local excision was performed. Because of a metastasis-suspect lymph node in the parotid gland, surgery included asservation of the sentinel lymph node, neck dissection and parotidectomy. A complete reconstruction of the ear was achieved using a bilobed flap from the retroauricular and neck region with acceptable cosmetic and functional results by one-step surgery. One micrometastasis was detected in the nuchal region but not in the marked sentinel lymph node. An adequate one-step ear reconstruction as described may also be performed to the benefit of patients with high-risk melanomas, allowing early adjuvant therapy.

Cytokine resistance is a well-established feature of melanoma cell progression and represents also a major obstacle in immunotherapy of patients with metastatic melanoma. To check whether suppressors of cytokine signalling (SOCS) play a role in cytokine resistance and tumor progression of melanoma, we investigated the expression and regulation of SOCS-1, an established negative regulator of interleukin-6 (IL-6) and interferon (IFN) signalling. In vitro SOCS-1 transcripts were detectable by RT-PCR in 8 out of 8 human melanoma cell lines derived from different tumor stages. Normal human melanocytes also expressed SOCS-1 mRNA in the presence or absence of artificial growth factors. Both IL-6 and alpha-IFN induced rapid and transient SOCS-1 mRNA expression in WM35 and WM9 melanoma cells. At the protein level, SOCS-1 was undetectable in normal human melanocytes whereas uniformly expressed in all tested melanoma cell lines. The aberrant SOCS-1 protein expression in melanoma cells was recapitalized in situ as shown by immunohistochemical analysis. SOCS-1 immunoreactivity was closely related to tumor invasion (Clark level), tumor thickness according to Breslow, and stage of the disease. In contrast, melanocytes in normal skin or melanocytic nevi lacked SOCS-1 protein expression. Our findings show that melanoma cells express a member of the SOCS family, SOCS-1, in vitro and in situ. SOCS-1 is a progression marker of human melanoma and may downregulate biological responses by endogenous and/or therapeutically administered cytokines.

The immune system plays an important role in the defense against malignant melanoma. Interferon-alpha (IFN-alpha) and interleukin-2 (IL-2) are therapeutically used for unspecific stimulation of the immune system. After intralesional injections of these cytokines into cutaneous melanoma metastases, regression has been observed. In view of its immunomodulating effects, imiquimod appears as an additional promising therapeutic option for treatment of malignant tumors. In a case report, we present combined therapy with intralesional IL-2, pegylated IFN-alpha-2b and topical imiquimod 5% cream for disseminated cutaneous metastatic malignant melanoma stage IIIa. This therapy achieved an almost complete remission. In a treatment period of eight months, side effects remained tolerable. Histologically, both fibrosis and inflammation were found in the regressing lesions. After the end of therapy, no disease progression occurred during 11 months follow-up.

Interleukin-2 (Il-2) is widely used for treatment of carcinomas, leukemia, and melanoma. Adverse drug effects of Il-2 include various systemic and generalized cutaneous drug reactions. In the following we report on a persistent inflammatory reaction at the injection site of Il-2. A patient received chemoimmuno-therapy for treatment of metastasizing melanoma, including subcutaneous application of recombinant interleukin-2 (Proleukin((R))). Within a few days reddish nodules developed at the injection sites that persisted after cessation of Il-2 injections for two years until final lethal outcome. Histologic examination revealed a lobular panniculitis expressing atypical lymphocytes and multinucleated histiocytes.

We report a 72-year-old male patient with a nodular malignant melanoma that was associated with focal suprabasal acantholysis (FSA). This phenomenon, which is regarded as an incidental finding by dermatopathologists, may be associated with inflammatory and also neoplastic skin diseases. Haematoxylin and eosin stained sections from an erythematous plaque surrounding the patient's tumour showed FSA, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) on normal human skin, monkey oesophagus and rat urinary bladder were negative. On electron microscopy few desmosomes could be detected in the basal cell layer of the acantholytic areas and there was a nearly complete loss of these structures in the spinous cell layer. Only remnants of cytoplasmic plaques and keratin filaments could be observed in those areas. In contrast, adherens junctions appeared to be well preserved. An enzyme-linked immunosorbent assay (ELISA) using recombinant fusion proteins as antigens did not show circulating autoantibodies against desmoglein 1 (Dsg1) or desmoglein 3 (Dsg3). In contrast, immunoblotting revealed autoantibodies directed against keratinocyte antigens with a molecular weight of 85 kDa and 250 kDa, the first band corresponding to the molecular weight of comigrating plakoglobin. Immunoprecipitation with patient serum also revealed a 85-kDa band. We conclude that these autoantibodies, probably in conjunction with cofactors produced by the tumour, could play a part in the pathogenesis of this variant of FSA, for which we propose the term 'localized paraneoplastic pemphigus.'

The origin of diffuse melanosis resulting from metastatic melanoma is unknown. We examined the light microscopic and ultrastructural changes in the skin of an affected 35-year-old woman and determined the peripheral blood levels of melanocyte growth factors. A total of 7 biopsy specimens were examined by light and electron microscopy and immunohistology (S-100, HMB45, MART1, CD68, MAC387). Serum/plasma levels of melanocyte growth factors of the patient were determined by enzyme-linked immunosorbent assays and compared with those of normal volunteers (n = 10) and amelanotic patients with metastatic melanoma (n = 10), matched to the UICC stage of the affected patient. Hyperpigmented but otherwise apparently normal skin of the patient displayed epidermal melanocyte hyperplasia, increased melanogenesis, and dermal pigment stored in histiocytes and other cells along with extracellular deposits. Blood levels of alpha-melanocyte stimulating hormone, hepatocyte growth factor, and endothelin-1 were significantly elevated in the affected patient. Aberrant production of these factors may not only be responsible for activation of the pigment system in diffuse melanosis of metastatic melanoma, but also for increased proliferation, motility, and pigment incontinence of normal and malignant melanocytes.

Xanthomatous changes are unusual lesions of the corpus endometrium that may mask endometrial carcinoma. The term "xanthomatous endometritis" refers to morphological changes frequently induced by estrogen stimulation. We report for the first time a case of uterine metastasis of balloon-cell melanoma mimicking xanthomatous endometritis. Light microscopic, immunohistological, and ultrastructural results are presented and discussed in connection with our ideas on the pathogenesis of this peculiar tumor. The findings favor the hypothesis of a regressive phenomenon in the balloon-cell transformation of melanoma cells. The melan-A immunohistology seems to be more important in the diagnosis of balloon cell melanoma than the classic melanoma antibody HMB 45.

Background and Objective: Malignant melanomas on sun-exposed skin are often poorly circumscribed and thus recur frequently. The aim of our clinical trial was to compare conventional to a modified micrographic surgery of primary melanoma., Patients/methods: 28 patients with in-situ (n = 7) and invasive (n = 21) melanoma were treated with conventional surgical excision with wide margins; eight of these patients developed a local recurrence. In comparison, 20 patients with primary in-situ (n = 7) and invasive (n = 13) melanoma as well as four patients with recurrence after conventional surgery underwent modified micrographic surgery with delayed closure of the wound., Results: Paraffin specimens of the margins revealed in half of the patients remnants of melanoma although excision was extended to non-lesional skin. Within a mean follow-up period of 21.3 months, none of our patients treated by modified surgery developed a recurrence., Conclusions: Local recurrencies of melanoma on sun exposed skin may be avoided by means of a modified micrographic surgery using permanent histologic sections.

Expression of mutant p53 detected by immunohistochemistry has been described in human malignant melanoma, but there are few reports of molecular analyses. To investigate the genetic basis for p53 expression in malignant melanoma, we examined 58 primary tumors and 5 cutaneous metastases. The entire coding sequence of the p53 gene was screened by single-strand conformation polymorphism analysis and direct genomic sequencing of polymerase chain reaction products. p53 and mdm-2 expression were studied by immunohistochemistry. Two p53 gene mutations could be found in 1/63 samples examined, both having occurred in the same specimen from a patient with a nodular melanoma. p53 and mdm-2 expression were found immunohistochemically to increase with tumor progression both in frequency and in the mean proportion of positive cells, with the same cases staining positively for both antibodies. Our results suggest that a) p53 gene mutations are a rare event in human melanoma; b) accumulation and thus immunohistochemically detectable expression of p53 may result from posttranslational mechanisms affecting the p53 gene product; and c) p53 and mdm-2 are more important in late events in melanoma carcinogenesis.

Background Uveal melanomas represent 3·1% of all melanomas, with a high potential of metastatic disease of up to 50%, where the median survival time is 6 months. Though liver metastases dominate as the primary site for metastasis, the existence of primary skin metastases is still under discussion but has been reported in only a few studies. Objectives We present two cases in which patients with a known history of uveal melanoma developed melanoma skin metastases. Methods Mutational analysis was performed to clarify the origin of the metastases (uvea or skin). Results The analyses revealed GNA11 mutations, which are typical for uveal melanoma. These cases strongly suggest the skin to be the primary site of uveal melanoma. Conclusions Knowledge about the mutational status of uveal melanomas opens the opportunity for future targeted therapies that directly interact with the mutation and its activated signal cascades. First trials in uveal melanoma have shown promising results with MEK inhibitors. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding a 23 year-old white woman, with a history of melanoma, who developed multiple melanomas following a self-injection of unlicensed melanotan-II.

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