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Background: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions., Objectives: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment., Methods: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets., Results: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAF WT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAF V600E compared with BRAF WT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAF WT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAF V600E expression in BRAF WT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAF WT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAF V600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages., Conclusions: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma., (© 2021 British Association of Dermatologists.)

Background: BRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes., Patients and Methods: The BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients' clinical characteristics. Kaplan-Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression., Results: A highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively)., Conclusion: These results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition., Competing Interests: Disclosure The authors have declared no conflicts of interest. Data sharing All data relevant to the study are included in the article or uploaded as online supplemental information. Individual patient data cannot be shared. Disclaimer The views expressed are those of the author(s) and not necessarily those of the Candiolo Cancer Institute or the University of Turin. Ethics approval Clinical-pathological data were recovered after obtaining the informed consent from all the patients, approved by the medical ethical committee of the FPO-IRCCS, and carried out according to the principles of the Declaration of Helsinki., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Malignant melanoma is the third most common type of tumor that causes brain metastases. Patients with cerebral involvement have a dismal prognosis and their treatment is an unmet medical need. Brain involvement is a multistep process involving several signaling pathways such as Janus kinase/signal Transducer and Activator of Transcription (JAK/STAT), Phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT), Vascular Endothelial Growth Factor and Phosphatase and Tensin Homolog (PTEN). Recently therapy that targets the MAPK signaling (BRAF/MEK inhibitors) and immunotherapy (anti-CTLA4 and anti-PD1 agents) have changed the therapeutic approaches to stage IV melanoma. In contrast, there are no solid data about patients with brain metastases, who are usually excluded from clinical trials. Retrospective data showed that BRAF-inhibitors, alone or in combination with MEK-inhibitors have interesting clinical activity in this setting. Prospective data about the combinations of BRAF/MEK inhibitors have been recently published, showing an improved overall response rate. Short intracranial disease control is still a challenge. Several attempts have been made in order to improve it with combinations between local and systemic therapies. Immunotherapy approaches seem to retain promising activity in the treatment of melanoma brain metastasis as showed by the results of clinical trials investigating the combination of anti-CTL4 (Ipilimumab) and anti-PD1(Nivolumab). Studies about the combination or the sequential approach of target therapy and immunotherapy are ongoing, with immature results. Several clinical trials are ongoing trying to explore new approaches in order to overcome tumor resistance. At this moment the correct therapeutic choices for melanoma with intracranial involvement is still a challenge and new strategies are needed.

BRAF signaling is involved in melanoma growth in more than half of metastatic patients. In the last few years, new drugs that block this pathway have significantly improved the outcomes of patients with metastatic melanoma. Ninety percent of BRAF mutations involve exon 15, and the most frequent, V600E, results from the amino acid change from valine (V) to glutamic acid (E). BRAF inhibitor treatments have shown a notable overall response rate and improvements in progression-free and overall survival. Rare BRAF mutations of codon 599 have been also described in a few patients with papillary thyroid cancer and melanoma. Nowadays, no evidence is available in the literature, describing the role of target therapies as treatment in patients with this specific codon mutation. We describe the case of a young woman with metastatic melanoma with a particular BRAF mutation, T599I, who has benefited from treatment with a BRAF inhibitor, vemurafenib.

Background: Sentinel lymph node (SLN)-positive melanoma patients are a heterogeneous group of patients with survival rates ranging from ∼20 to over 80%. No data are reported concerning the role of histological regression on survival in stage III melanoma., Methods: The study included 365 patients with positive SLN from two distinct hospitals. The model was developed on patients from 'AOU Città della Salute e della Scienza di Torino', and externally validated on patients from IRCCS of Candiolo. Survival analyses were carried out according to the presence of regression and adjusted for all other prognostic factors., Results: Among patients followed at 'AOU Città della Salute e della Scienza di Torino' (n=264), the median follow-up time to death or censoring (whatever two events occurred earlier) was 2.7 years since diagnosis (interquartile range: 1.3-5.8). In all, 79 patients died from melanoma and 11 from other causes. Histological regression (n=43) was associated with a better prognosis (sub-HR=0.34, CI 0.12-0.92), whereas the other factors above showed an inverse association. In the external validation, the concordance index was 0.97 at 1 year and decreased to 0.66 at 3 years and to 0.59 at 5 years. Adding histological regression in the prognostic model increased the discriminative ability to 0.75 at 3 years and to 0.62 at 5 years. Finally, using a cutoff of 20% for the risk of death led to a net re-classification improvement of 15 and 11% at 3 and 5 years after diagnosis, respectively., Conclusions: Histological regression could lead to an improvement in prognostic prediction in patients with stage III-positive SLN melanoma.

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4 Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP + mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP + mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP + MCs (1/42) compared with the eGFP - counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells ( n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP + mCSCs ( P = 0.001). Sequential CHT-immunotherapy treatment retained antitumor activity ( n = 12, P = 0.001) reducing mCSC rates ( P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277-88. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Purpose: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC)., Experimental Design: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4., Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11-117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8)., Conclusions: For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer., (©2013 AACR.)

Unlabelled: AIMS, PATIENTS AND METHODS: The umbilical melanoma is rare, and the surgical treatment can create difficulties for both radical excision and plastic reconstruction. Our aims are to present a case of primary melanoma of the umbilicus and to discuss the best surgical treatment, as well as review the relevant literature., Results: Surgical excision of primary melanoma of the umbilicus must be carried out to reach the peritoneum. Sentinel lymph node biopsy must be carried as well as plastic reconstruction., Conclusion: Despite the progress in new medical therapy for melanoma, suitable surgical excision is, at present, the only treatment able to improve patient prognosis. In this report we describe the surgical treatment and plastic reconstruction of a case of umbilical melanoma., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

BRAF(V600E) mutation has been frequently reported in different types of melanocytic lesions, but its role in melanomagenesis is poorly understood, having been associated with either the proliferative-induced MAPK pathway activation or the acquisition of oncogene-driven senescence. The presence of BRAF alterations has been related to sun exposure, although the molecular mechanisms underlying this event are only partly known. To elucidate the relationships among BRAF/NRAS alterations, MAPK pathway activation, and sun exposure, we examined 22 acquired nevi and 18 cutaneus melanomas from 38 patients. Microdissected tissues from each lesion were subjected to BRAF/NRAS mutation analysis by sequencing, allele-specific PCR and pyrosequencing assay. The same lesions were also examined for the expression of phosphorylated ERK1/2. Phototype and an accurate history of sun exposure were evaluated for each patient. BRAF(V600E) mutation was detected in 50% of the acquired nevi and in 70% of the cutaneus melanomas in the absence of NRAS alterations. The fraction of alleles carrying BRAF(V600E) substitution was variable but strongly associated with sun exposure. In contrast, no relationship was evidenced between the presence of this mutation and patients' phototype, phosphorylated ERK1/2 expression, or Clark's level. Our findings indicate that in melanocytic lesions, BRAF(V600E) mutation can affect a subset of the cells and is associated with the type and quantity of sun exposure. This mutation is independent of the nevo-melanoma progression and unrelated to ERK phosphorylation, suggesting that alternative mechanisms to the MAPK activation are also involved in this type of transformation.

Clear cell sarcoma of the tendons and aponeuroses is an aggressive, rare soft tissue tumour that occurs predominantly in the extremities of young adults. Although it appears to be histogenetically related to melanoma, its clinical behaviour resembles soft tissue sarcoma. Prognosis is reported to be poor due to the great propensity of regional and distant metastases. The risk of metastases to regional lymph nodes is very high. We describe a case of clear cell sarcoma of the hand and evaluate the feasibility of the sentinel lymph node biopsy (SLNB) technique in this kind of tumour. Up to now, there are no reports that describe the use of SLNB in clear cell sarcoma. The SLNB technique was carried out with success and the sentinel lymphnode was easily identified. Histological examination of the lymphnode allowed identification of metastatic cells and thus provide for radical lymphadenectomy. We maintain that SLNB can be successfully used for this type of tumour although this result should be evaluated and confirmed by larger case studies.

Background: Current treatment for melanoma of the lower limb includes excision of the primary tumor with ilioinguinal lymphadenectomy in the case of lymph node metastases. The standard surgical approach includes sectioning of the inguinal ligament to gain access to the iliac nodes. More recently, some authors have reported that extraperitoneal laparoscopically assisted ilioinguinal lymphadenectomy for the treatment of malignant melanoma is feasible and less aggressive than standard open surgery. So far, no publications have described transperitoneal laparoscopic iliac lymphadenectomy (TPLND)., Methods: From November 2001 to June 2002, 13 patients with ilioinguinal node melanoma metastases underwent TPLND (stage IIIA in 1 case, IIIB in 5 cases, IIIC in 4 cases, and IV in 3 cases)., Results: In all 13 cases, the TPLND and groin dissection was performed correctly. Operative time, intra- and postoperative complications, number of lymph nodes retrieved, immediate morbidity, hospital stay, and feasibility of TPLND were evaluated., Conclusions: This study was conducted to evaluate the feasibility and the preliminary results of TPLND used to manage malignant melanoma of the lower limb. This approach has many advantages over the traditional procedure: less surgical trauma, no incision of the abdominal muscles or the inguinal ligament, and less postoperative pain. Moreover, as compared with extraperitoneal laparoscopically assisted ilioinguinal lymphoadenectomy, it provides an improved view of the operative area, dissection zone, and surrounding structures. Further research is needed to confirm these preliminary results regarding the potential applications of this method for treating malignant metastasis to the lower limb.

Background: Cutaneous metastasis from neuroendocrine carcinomas of visceral origin is rarely described in indexed literature. The primary sites of origin include: lung (Wick et al., J Am Acad Dermatol 1985; 13: 134), larynx (Zambruno et al., Ann Dermatol Venereol 1989; 116: 855; Schmidt et al., J Laryngol Otol 1994; 108: 272; Guerzider et al., Ann Pathol 1991; 11 (4): 253), mediastinum (Yoshimasu et al., J Dermatol 2001; 28 (3): 168), uterus (Fogaca et al., J Cutan Pathol 1993; 20: 455), and thymus (Wick et al., J Am Acad Dermatol 1985; 13: 134)., Methods: In this report, the authors present the clinical, histological, immunohistochemical, and ultrastructural characteristics of secondary skin localizations of a neuroendocrine laryngeal tumor that occurred in a 61-year-old man. The complete follow up of the case is described and a brief revision of the terminology and classification of neuroendocrine neoplasms of the larynx is discussed, since a significant relationship exists between the degree of differentiation and biological behavior., Results: On histological examination, the secondary cutaneous localization appeared to be more dedifferentiated compared to the primary tumor. The immunohistochemical patterns of reactivity were similar in both neoplasms, showing expression of neuroendocrine and epithelial markers., Conclusions: An important issue of prognostic significance is to differentiate a cutaneous metastasis of a neuroendocrine carcinoma from the primary small cell-undifferentiated carcinoma of the skin (Merkel cell carcinoma).

Here we report a case of Pyoderma gangrenosum (PG) associated with Crohn's disease successfully treated with infliximab. The efficacy of this drug in many inflammatory diseases has already been reported, but its use in PG has only been seen in very few cases. Our study confirms that this therapy is a valid alternative solution for treating PG, which is often unresponsive to conventional therapies.

The mechanisms of extracorporeal photochemotherapy (ExP) therapeutic activity in cutaneous T-cell lymphomas (CTCLs) are not yet well understood, even though it has been suggested that a major mechanism may be induction of apoptosis. In vitro studies demonstrate that UVA-induced apoptosis is mediated by CD95-Fas expression and inhibited by Bcl-2 up-regulation and that UVA irradiation is able to down-regulate Bcl-2 expression. High-resolution multiparameter flow-cytometric analyses were used to evaluate Bcl-2/CD95-Fas expression on phenotypically identifiable circulating clonal T cells from 7 patients with CTCL (4 with Sézary syndrome and 3 with mycosis fungoides with peripheral involvement) before and during ExP, in an attempt to ascertain whether Bcl-2/CD95-Fas status can be related to the hematologic response. A Bcl-2 normal phenotype before ExP or a normalization in Bcl-2 expression during ExP were related to a better clinical response, whereas a persistent Bcl-2 high expression was a negative prognostic factor. On the other hand, no response was found in patients with a CD95-Fas-negative phenotype, whereas the expression of CD95-Fas was associated with hematologic remission. Although further studies are needed to confirm these preliminary results, this study suggests that Bcl-2 and CD95-Fas expression could be evaluated, together with the other known clinical and immunologic factors, as additional parameters related to clinical response in patients with CTCL undergoing ExP.

Background: Malignant melanoma has an unpredictable biologic behavior and is the neoplasm with the greatest propensity for cardiac involvement. Although relatively frequent at autopsy, cardiac metastases are rarely identified antemortem., Methods: We reviewed 2,810 patients with histologically confirmed malignant melanoma, who were diagnosed and followed up by our clinic. Clinical, histological, and imaging data are presented., Results: Five cases of metastatic melanoma of the heart were identified out of 314 melanoma patients with visceral involvement. One case of a 53-year-old woman, who died unexpectedly during her first chemotherapy course, is described in detail. Postmortem examination determined the cause of death to be the presence of multiple melanoma metastases in the heart, even though the patient had shown no signs of cardiac involvement., Conclusions: The unpredictable biologic behavior of melanoma may lead to unusual metastatic sites, and, therefore, the heart also should be included in routine examinations., (Copyright 2000 Wiley-Liss, Inc.)

Objective: To measure nitric oxide (NO) concentration in exhaled air of patients with systemic sclerosis (SSc) and to investigate its relationships with lung involvement, complicated or not by pulmonary hypertension (PH)., Methods: Exhaled NO was measured by chemiluminescence in 47 patients with SSc (16 with PH) and in 30 controls. All the patients underwent Doppler echocardiography to assess pulmonary artery pressure (PAP), lung function tests, and thin section computed tomographic scans of the lung to quantify the extent of fibrosing alveolitis., Results: Exhaled NO levels were higher in patients with SSc (16.6 +/- 9.1 ppb), particularly those with interstitial lung disease (ILD) (18.3 +/- 10.4 ppb), compared to controls (9.9 +/- 2.9 ppb; p < 0.0001). In patients with PH, exhaled NO was less than in patients without PH (10.7 +/- 5.9 vs 19.6 +/- 9 ppb, respectively; p < 0.001), and patients with PH without ILD had even lower exhaled NO than patients with PH and ILD (6.6 +/- 1.1 vs 12.6 +/- 6.3 ppb; p = 0.004). There was an inverse correlation between PAP and exhaled NO (r = 04).53, p = 0.004). Exhaled NO was not correlated to age, disease duration, current therapy, or form of disease (limited or diffuse)., Conclusion: The increased concentration of exhaled NO in patients with SSc may reflect respiratory tract inflammation. The relatively low value of exhaled NO in patients with PH and the negative correlation between PAP and exhaled NO suggest the important role of NO in regulating pulmonary vascular resistance in patients with SSc.

Background: Mycosis fungoides (MF) is rare in young patients. Its clinical behavior is still uncertain, as some reports have suggested that it has a more aggressive course than does the adult-onset type., Aim: To ascertain if early-onset MF represents a heterogeneous group of cutaneous T cell lymphomas., Materials and Methods: Clinical, immunohistopathological and follow-up data of early-onset (<20 years of age) MF cases reported in the literature (n = 42) plus 7 described herein were compared with those of a cohort of adult-onset MF patients (n = 252) diagnosed at our institution since 1975., Results: The majority of the 49 early-onset MF patients had patch-plaque stage disease at diagnosis. Ten had hypopigmented lesions. The predominant phenotype was CD3+ CD4+CD7-CD8-. Seven patients had a stage progression, 6 with extracutaneous involvement. Five- and 10-year survival rates were 93 and 74%, respectively., Conclusions: No statistically significant differences were found in the disease course between early- and adult-onset MF.