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Next-generation sequencing (NGS) is increasingly being utilized as an ancillary tool for diagnostically challenging melanocytic neoplasms. It is incumbent upon the pathology community to perform studies assessing the benefits and limitations of these tools in specific diagnostic scenarios. One of the most challenging diagnostic scenarios faced by skin pathologists involves accurate diagnosis of desmoplastic melanocytic neoplasms (DMNs). In this study, 20 expert melanoma pathologists rendered a diagnosis on 47 DMNs based on hematoxylin and eosin sections with demographic information. After submitting their diagnosis, the experts were given the same cases, but this time with comprehensive genomic sequencing results, and asked to render a diagnosis again. Identification of desmoplastic melanoma (DM) improved by 7%, and this difference was statistically significant ( P <0.05). In addition, among the 15 melanoma cases, in the pregenomic assessment, only 12 were favored to be DM by the experts, while after genomics, this improved to 14 of the cases being favored to be DM. In fact, some cases resulting in metastatic disease had a substantial increase in the number of experts recognizing them as DM after genomics. The impact of the genomic findings was less dramatic among benign and intermediate-grade desmoplastic tumors (BIDTs). Interobserver agreement also improved, with the Fleiss multirater Kappa being 0.36 before genomics to 0.4 after genomics. NGS has the potential to improve diagnostic accuracy in the assessment of desmoplastic melanocytic tumors. The degree of improvement will be most substantial among pathologists with some background and experience in bioinformatics and melanoma genetics., Competing Interests: Conflicts of Interest and Source of Funding: P.G. has served as a consultant for Castle Biosciences and has received an honorarium for this, he has also received royalties for textbooks from Elsevier. For the remaining authors, none were declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Drivers of Spitz neoplasms include activating point mutations in HRAS and Spitz-associated genomic fusions. It has become evident that some BRAF -mutated melanocytic neoplasms can morphologically mimic Spitz tumors (STs). These have been termed BRAF mutated and morphologically spitzoid (BAMS). In this study, 17 experts from the International Melanoma Pathology Study Group assessed 54 cases which included 40 BAMS and 14 true STs. The participants reviewed the cases blinded to the genomic data and selected among several diagnostic options, including BAMS, ST, melanoma, and other. A total of 38% of all diagnostic selections in the BAMS cases were for BAMS, whereas 32% were for ST. In 22 of the BAMS cases, the favored diagnosis was BAMS, whereas in 17 of the BAMS cases, the favored diagnosis was ST. Among the 20 cases in the total group of 54 with the highest number of votes for ST, half were BAMS. Of BAMS, 75% had a number of votes for ST that was within the SD of votes for ST seen among true ST cases. There was poor interobserver agreement for the precise diagnosis of the BAMS (kappa = 0.16) but good agreement that these cases were not melanoma (kappa = 0.7). BAMS nevi/tumors can closely mimic Spitz neoplasms. Expert melanoma pathologists in this study favored a diagnosis of ST in nearly half of the BAMS cases. There are BAMS cases that even experts cannot morphologically distinguish from true Spitz neoplasms., Competing Interests: Conflicts of Interest and Source of Funding: P.G. has served as a consultant for Castle Biosciences and has received royalties for textbooks from Elsevier. For the remaining authors, none were declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Fusion genes involving homologs of protein kinase C (PKC) have been identified in a variety of tumors. We report the clinical and histologic presentation of 51 cutaneous melanocytic neoplasms with a PKC fusion gene (involving PRKCA in 35 cases, PRKCB in 15 cases, and PRKCG in a single case). Most tumors were in young adults (median age, 29.5 years; range, 1-73 years) but some presented in newborns. Histologically, 42 tumors were classified as benign, presenting predominantly as biphasic dermal proliferation (88%) with nests of small melanocytes surrounded by fibrosis with haphazardly arranged spindled and dendritic melanocytes, resembling those reported as "combined blue nevi." Most tumors (60%) were heavily pigmented and in 15%, hyperpigmented epithelioid melanocytes were present at the dermoepidermal junction. Two lesions were paucicellular and showed marked sclerosis. Three tumors, including 2 proliferating nodules, were considered intermediate grade. Six tumors had sheets of atypical melanocytes infiltrating the dermis and were classified as melanomas. Two of the melanomas displayed loss of BAP1 nuclear expression. The median follow-up time was 12 months, with 1 patient alive with metastatic disease and 1 dying of their melanoma. These results suggest that melanocytic tumors with PKC fusion genes have characteristic histopathologic features, which are more similar to blue nevi than to pigmented epithelioid melanocytomas. As is the case with GNA-mutated blue nevi, they can progress to melanomas via BAP1 inactivation and metastasize., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Importance: A standardized pathology classification system for melanocytic lesions is needed to aid both pathologists and clinicians in cataloging currently existing diverse terminologies and in the diagnosis and treatment of patients. The Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis (MPATH-Dx) has been developed for this purpose., Objective: To revise the MPATH-Dx version 1.0 classification tool, using feedback from dermatopathologists participating in the National Institutes of Health-funded Reducing Errors in Melanocytic Interpretations (REMI) Study and from members of the International Melanoma Pathology Study Group (IMPSG)., Evidence Review: Practicing dermatopathologists recruited from 40 US states participated in the 2-year REMI study and provided feedback on the MPATH-Dx version 1.0 tool. Independently, member dermatopathologists participating in an IMPSG workshop dedicated to the MPATH-Dx schema provided additional input for refining the MPATH-Dx tool. A reference panel of 3 dermatopathologists, the original authors of the MPATH-Dx version 1.0 tool, integrated all feedback into an updated and refined MPATH-Dx version 2.0., Findings: The new MPATH-Dx version 2.0 schema simplifies the original 5-class hierarchy into 4 classes to improve diagnostic concordance and to provide more explicit guidance in the treatment of patients. This new version also has clearly defined histopathological criteria for classification of classes I and II lesions; has specific provisions for the most frequently encountered low-cumulative sun damage pathway of melanoma progression, as well as other, less common World Health Organization pathways to melanoma; provides guidance for classifying intermediate class II tumors vs melanoma; and recognizes a subset of pT1a melanomas with very low risk and possible eventual reclassification as neoplasms lacking criteria for melanoma., Conclusions and Relevance: The implementation of the newly revised MPATH-Dx version 2.0 schema into clinical practice is anticipated to provide a robust tool and adjunct for standardized diagnostic reporting of melanocytic lesions and management of patients to the benefit of both health care practitioners and patients.

Aims: The aim of this multicentre study was to harmonize programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) and melanoma scoring. To provide a reference for PD-L1 expression independently of the IHC protocol, PD-L1 mRNA expression was compared with IHC., Methods and Results: Standardized PD-L1 assays (22C3, 28-8, SP142, SP263) and laboratory-developed tests (QR1, 22C3) were evaluated on three IHC platforms with a training set (seven cases). mRNA expression was determined by RNAscope (CD274/PD-L1 probe) and analysed with image analysis. PD-L1 IHC findings were scored by seven blinded pathologists using the tumour proportion score (TPS), the combined positive score (CPS), and the MELscore. This method was validated by three blinded pathologists on 40 metastatic melanomas. Concordances among various antibody/platforms were high across antibodies [intraclass correlation coefficient (ICC) >0.80 for the CPS], except for SP142. Two levels of immunostaining intensity were observed: high (QR1 and SP263) and low (28-8, 22C3, and SP142). Reproducibilities across pathologists were higher for QR1 and SP263 (ICC ≥0.87 and ICC ≥0.85 for the TPS and the CPS, respectively). QR1, SP263 and 28-8 showed the highest concordance with mRNA expression. We developed a standardized method for PD-L1 immunodetection and scoring, tested on 40 metastatic melanomas. Concordances among antibodies were excellent for all criteria, and concordances among pathologists were better for the MELscore than for other scores., Conclusion: Harmonization of PD-L1 staining and scoring in melanomas with good concordance is achievable with the PD-L1 IHC protocols applied to other cancers; this reproducible approach can simplify daily practice., (© 2022 John Wiley & Sons Ltd.)

Spitz neoplasms, according to 2018 WHO Blue Book, are morphologically defined by spindled and/or epithelioid melanocytes and genetically by either HRAS mutations or kinase gene fusions. The terminology "spitzoid" refers to lesions with similar morphology but with alternate or undefined genetic anomalies. Herein, we present 3 melanocytic neoplasms with a spitzoid cytomorphology, variable nuclear atypia, and harboring undescribed fusions involving RASGRF1. Two cases presented as unpigmented papules on the heel of a 26-year-old female (case 1) and the forearm of a 13-year-old boy (case 2). They were classified as low-grade melanocytomas (WHO 2018). The third case appeared as a pigmented ulcer on the sole of a 72-year-old female (case 3) that displayed diagnostic features of an invasive melanoma (Breslow thickness 6 mm, Clark level V). A wide skin reexcision identified an epidermotropic metastasis, and sentinel lymph node biopsy displayed multiple subcapsular metastatic deposits. RNA sequencing revealed CD63::RASGRF1, EHBP1::RASGRF1, and ABCC2::RASGRF1 fusions in cases 1 to 3, respectively. They were confirmed by a RASGRF1 break-apart fluorescence in situ hybridization technique. Translocations of RASGRF1, a gene coding a guanine nucleotide exchange factor but not a kinase, have rarely been reported in tumors. While all these cases showed spitzoid cytomorphology, it is too early to tell if they are true Spitz neoplasms as currently defined., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The histopathological assessment of diagnostically ambiguous melanocytic proliferation remains one of the biggest challenges in the dermatopathology field. Preferentially expressed Antigen in MElanoma (PRAME) immunostaining has been shown highly specific for distinguishing unequivocal malignant melanocytic proliferation from benign ones. Knowledge on its utility for evaluating ambiguous melanocytic neoplasms remains limited. We retrieved in our institutional database all cases of diagnostically ambiguous melanocytic neoplasms from January 2016 to January 2021. Each case was subclassified into "favor benign" or "favor malignant" neoplasm using all collected data. Immunohistochemical expression of PRAME was assessed and correlated with the final subclassification. Using a previously proposed scoring system, diffuse immunopositivity (>75% of tumor cells) was considered positive. Furthermore, for ambiguous melanocytic proliferation occurring on a pre-existing nevus, the staining was considered positive if more than 75% of the morphologically atypical neoplastic cells were labeled, excluding morphologically unambiguous benign nevocytes. Fifty-five cases of ambiguous melanocytic proliferation were examined. Thirty-one cases were finally subclassified as "favor malignant" neoplasms and 24 as "favor benign" neoplasms. Thirty-one tumors showed immunopositivity for PRAME, representing, respectively, 8.3% and 93.5% of "favor benign" and "favor malignant" neoplasms. The specificity and sensitivity of PRAME immunohistochemistry for benign/malignant distinction were, respectively, 91.7% and 93.5%.PRAME IHC shows high sensitivity and specificity for distinguishing malignant challenging melanocytic proliferations from benign ones and could be used as an everyday tool. However, PRAME immunoreactivity should be interpreted cautiously, knowing that rare benign melanocytic neoplasms could show diffuse positivity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: The efficacy of immunotherapies in metastatic melanoma depends on a robust T cell infiltration. Oncogenic alterations of tumor cells have been associated to T cell exclusion. Identifying novel cancer cell-intrinsic non-genetic mechanisms of immune escape, the targeting of which would reinstate T cell recruitment, would allow to restore the response to anti-programmed cell death protein 1 (PD-1) antibody therapy. The epithelial-to-mesenchymal transition (EMT)-inducing transcription factor ZEB1 is a major regulator of melanoma cell plasticity, driving resistance to mitogen-activated protein kinase (MAPK) targeted therapies. We thus wondered whether ZEB1 signaling in melanoma cells may promote immune evasion and resistance to immunotherapy., Methods: We evaluated the putative correlation between ZEB1 expression in melanoma cells and the composition of the immune infiltrate in a cohort of 60 human melanoma samples by combining transcriptomic (RNA-sequencing) and seven-color spatial multi-immunofluorescence analyses. Algorithm-based spatial reconstitution of tumors allowed the quantification of CD8 + , CD4 + T cells number and their activation state (PD-1, Ki67). ZEB1 gain-of-function or loss-of-function approaches were then implemented in syngeneic melanoma mouse models, followed by monitoring of tumor growth, quantification of immune cell populations frequency and function by flow cytometry, cytokines secretion by multiplex analyses. Chromatin-immunoprecipitation was used to demonstrate the direct binding of this transcription factor on the promoters of cytokine-encoding genes. Finally, the sensitivity to anti-PD-1 antibody therapy upon ZEB1 gain-of-function or loss-of-function was evaluated., Results: Combined spatial and transcriptomic analyses of the immune infiltrates in human melanoma samples demonstrated that ZEB1 expression in melanoma cells is associated with decreased CD8 + T cell infiltration, independently of β-catenin pathway activation. ZEB1 ectopic expression in melanoma cells impairs CD8 + T cell recruitment in syngeneic mouse models, resulting in tumor immune evasion and resistance to immune checkpoint blockade. Mechanistically, we demonstrate that ZEB1 directly represses the secretion of T cell-attracting chemokines, including CXCL10. Finally, Zeb1 knock-out, by promoting CD8 + T cell infiltration, synergizes with anti-PD-1 antibody therapy in promoting tumor regression., Conclusions: We identify the ZEB1 transcription factor as a key determinant of melanoma immune escape, highlighting a previously unknown therapeutic target to increase efficacy of immunotherapy in melanoma., Trial Registration Number: NCT02828202., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Multiple BRCA1-associated protein 1 (BAP1)-inactivated melanocytic tumors (BIMTs) have been associated with a familial cancer syndrome involving germline mutations in BAP1., Objectives: We sought to describe the clinical and dermoscopic features of BIMTs., Methods: This was a retrospective, multicenter, case-control study. Participating centers contributed clinical data, dermoscopic images, and histopathologic data of biopsy-proven BIMTs. We compared the dermoscopic features between BIMTs and control patients., Results: The dataset consisted of 48 BIMTs from 31 patients (22 women; median age 37 years) and 80 control patients. Eleven patients had a BAP1 germline mutation. Clinically, most BIMTs presented as pink, dome-shaped papules (n = 24). Dermoscopically, we identified 5 patterns: structureless pink-to-tan with irregular eccentric dots/globules (n = 14, 29.8%); structureless pink-to-tan with peripheral vessels (n = 10, 21.3%); structureless pink-to-tan (n = 7, 14.9%); a network with raised, structureless, pink-to-tan areas (n = 7, 14.9%); and globular pattern (n = 4, 8.5%). The structureless with eccentric dots/globules pattern and network with raised structureless areas pattern were only identified in BIMT and were more common in patients with BAP1 germline mutations (P < .0001 and P = .001, respectively)., Limitations: Limitations included our small sample size, retrospective design, the absence of germline genetic testing in all patients, and inclusion bias toward more atypical-looking BIMTs., Conclusions: Dome-shaped papules with pink-to-tan structureless areas and peripheral irregular dots/globules or network should raise the clinical suspicion for BIMT., (Copyright © 2018 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Activating NTRK1 fusions have been described as oncogenic events across the spectrum of Spitz tumors. Herein we report a series of 38 Spitz tumors with NTRK1 fusion. These Spitz tumors have distinctive histopathologic features characterized by filigree-like rete ridges which are elongated, thin and branched, dermal melanocytes arranged in a rosette-like configuration, and marked diminishment of melanocyte size with descent into the dermis. These features are distinct from those of other genetically defined subtypes of Spitz tumors and can aid in microscopic diagnosis and help prioritize in case selection for molecular testing in the rare patients that need targeted therapy.

Recent advances in genomics have improved the molecular classification of cutaneous melanocytic tumors. Among them, deep penetrating nevi (DPN) and plexiform nevi have been linked to joint activation of the MAP kinase and dysregulation of the β-catenin pathways. Immunohistochemical studies have confirmed cytoplasmic and nuclear expression of β-catenin and its downstream effector cyclin D1 in these tumors. We assessed nuclear β-catenin immunohistochemical expression in a large group of DPN as well as in the four most frequent differential diagnoses of DPN: "blue" melanocytic tumors, Spitz tumors, nevoid and SSM melanomas, and pigmented epithelioid melanocytomas (PEM). Nuclear β-catenin expression was positive in 98/100 DPN and 2/16 of melanomas (one SSM and one nevoid melanoma with a plexiform clone) and was negative in all 30 Spitz, 26 blue, and 6 PEM lesions. In 41% DPN, β-catenin expression was positive in more than 30% nuclei. No differences were observed in cytoplasmic and nuclear cyclin D1 expression between these tumor groups, suggesting alternate, β-catenin-independent, activation pathways. We have subsequently studied nuclear β-catenin expression in a set of 13 tumors with an ambiguous diagnosis, for which DPN was part of the differential diagnosis. The three out of four patients showing canonical DPN mutation profiles were the only β-catenin-positive cases. We conclude that nuclear β-catenin expression, independently from CCND1 expression, in a dermal melanocytic tumor is an argument for its classification as DPN. In ambiguous cases and in early combined DPN lesions, this antibody can be helpful as a screening tool. β-Catenin is also potentially expressed in a subset of malignant melanomas with CTNNB1 mutations.

Due to its high proclivity to metastasize, and despite the recent development of targeted and immune therapy strategies, melanoma is still the deadliest form of skin cancer. Therefore, understanding the molecular mechanisms underlying melanoma invasion remains crucial. We previously characterized Tspan8 for its ability to prompt melanoma cell detachment from their microenvironment and trigger melanoma cell invasiveness, but the signaling events by which Tspan8 regulates the invasion process still remain unknown. Here, we demonstrated that β-catenin stabilization is a molecular signal subsequent to the onset of Tspan8 expression, and that, in turn, β-catenin triggers the direct transcriptional activation of Tspan8 expression, leading to melanoma invasion. Moreover, we showed that β-catenin activation systematically correlates with a high expression of Tspan8 protein in melanoma lesions from transgenic Nras; bcat* mice, as well as in deep penetrating naevi, a type of human pre-melanoma neoplasm characterized by a combined activation of β-catenin and MAP kinase signaling. Overall, our data suggest that β-catenin and Tspan8 are part of a positive feedback loop, which sustains a high Tspan8 expression level, conferring to melanoma cells the invasive properties required for tumor progression and dissemination.

Ectodysplasin receptor EDAR is seen as a typical Tumor Necrosis Factor receptor (TNFR) family member known to interact with its ligand Eda-A1, and signaling mainly through the nuclear factor-kappaB (NF-κB) and c-jun N-terminal kinases pathways. Mutations in genes that encode proteins involved in EDAR transduction cascade cause anhidrotic ectodermal dysplasia. Here, we report an unexpected pro-apoptotic activity of EDAR when unbound to its ligand Eda-A1, which is independent of NF-κB pathway. Contrarily to other death receptors, EDAR does recruit caspase-8 to trigger apoptosis but solely upon ligand withdrawal, thereby behaving as the so-called dependence receptors. We propose that pro-apoptotic activity of unbound EDAR confers it a tumor suppressive activity. Along this line, we identified loss-of-pro-apoptotic function mutations in EDAR gene in human melanoma. Moreover, we show that the invalidation of EDAR in mice promotes melanoma progression in a B-Raf mutant background. Together, these data support the view that EDAR constrains melanoma progression by acting as a dependence receptor.

Primary malignant melanoma of the esophagus is rare, accounting for less than 0.1-0.2% of all esophageal malignancies. It is associated with a poor outcome due to late detection and high metastatic potential. Here, we report a case of esophageal cancer, which was initially diagnosed as an adenocarcinoma and finally was confirmed as a primary malignant melanoma. This 75-year-old Caucasian male had a history of dysphagia and recent lingering abdominal pain. First biopsy showed a poorly-differentiated adenocarcinoma. He was then treated with neoadjuvant radiochemotherapy. Biopsies were repeated because of an incomplete tumor response, evaluated by endoscopic and imaging studies. The final diagnosis was a malignant melanoma. The patient has been treated with immune-checkpoint inhibitor, nivolumab, an anti-PD1 antibody.

Uveal melanoma (UM) is the most common primary tumor in the adult, and disseminates to the liver in half of patients. A 15-gene expression profile prognostic assay allows to determine the likelihood of metastasis in patients using their ocular tumor DNA, but a cure still remains to be discovered. The serotonin receptor 2B represents the discriminant gene of this molecular signature with the greatest impact on the prognosis of UM. However, its contribution to the metastatic potential of UM remains unexplored. The purpose of this study was to investigate the effects of a selective serotonin receptor 2B antagonist on cellular and molecular behaviours of UM cells. UM cell lines expressing high level of serotonin receptor 2B proteins were selected by Western blotting. The selective serotonin receptor 2B antagonist PRX-08066 was evaluated for its impact on UM cells using viability assays, phosphorylated histone H3 immunostainings, clonogenic assays, migration assays, invasion assays and membrane-based protein kinase phosphorylation antibody arrays. The pharmacological inhibition of the serotonin receptor 2B reduced the viability of UM cells and the population in mitosis, and impaired their clonogenicity and potential of migration. It also decreased the phosphorylation of kinases from signaling pathways classically activated by the serotonin receptor 2B, as well as kinases β-catenin, Proline-rich tyrosine kinase 2, and Signal transducer and activator of transcription 5. Our findings support a role for the serotonin receptor 2B in the proliferation and migration of UM cells, through activation of many signaling pathways such as WNT, Focal adhesion kinase and Janus kinase/STAT.

We report the case of a 57-year-old man, who is hospitalized for the surgery of a gallbladder mass associated by an increase in fluorodeoxyglucose-activity on positron emission tomography/computed tomography scan. This is an incidental finding occurring during monitoring of a skin melanoma. A cholecystectomy is performed. Microscopic examination identified an infiltration of the gallbladder wall by a proliferation of atypical pigmented spindled melanocytes with numerous mitoses. The immunohistochemical analysis confirmed the melanocytic nature of this proliferation with the staining of HMB-45, S100 protein and Melan-A. A complementary immunohistochemical (p16, desmin and BRAFV600E) and molecular (BRAF sequencing) study is performed. The results are consistent with the hypothesis of a gallbladder metastasis of a cutaneous melanoma is proposed. Gallbladder metastases of melanoma are exceptional. The aim of our work is to describe a new case with immunohistochemical and molecular characterization, to review the literature on this topic and to consider the main differential diagnosis (primary malignant melanoma of the gallbladder)., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Melanoma is well known for its propensity for lethal metastasis and resistance to most current therapies. Tumor progression and drug resistance depend to a large extent on the interplay between tumor cells and the surrounding matrix. We previously identified Tetraspanin 8 (Tspan8) as a critical mediator of melanoma invasion, whose expression is absent in healthy skin. The present study investigated whether Tspan8 may influence cell-matrix anchorage and regulate downstream molecular pathways leading to an aggressive behavior. Using silencing and ectopic expression strategies, we showed that Tspan8-mediated invasion of melanoma cells resulted from defects in cell-matrix anchorage by interacting with β1 integrins and by interfering with their clustering, without affecting their surface or global expression levels. These effects were associated with impaired phosphorylation of integrin-linked kinase (ILK) and its downstream target Akt-S473, but not FAK. Specific blockade of Akt or ILK activity strongly affected cell-matrix adhesion. Moreover, expression of a dominant-negative form of ILK reduced β1 integrin clustering and cell-matrix adhesion. Finally, we observed a tumor-promoting effect of Tspan8 in vivo and a mutually exclusive expression pattern between Tspan8 and phosphorylated ILK in melanoma xenografts and human melanocytic lesions. Altogether, the in vitro, in vivo and in situ data highlight a novel regulatory role for Tspan8 in melanoma progression by modulating cell-matrix interactions through β1 integrin-ILK axis and establish Tspan8 as a negative regulator of ILK activity. These findings emphasize the importance of targeting Tspan8 as a means of switching from low- to firm-adhesive states, mandatory to prevent tumor dissemination.

Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3β, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.

Importance: The differential diagnosis between proliferative nodules (PNs) and melanoma arising in congenital melanocytic nevi (CMN) is crucial, as patients with PNs most often experience no increased risk of melanoma with metastases and death., Objective: To analyze the utility of immunohistochemistry and fluorescence in situ hybridization (FISH) in distinguishing PNs from childhood and adult-onset melanoma arising in CMN., Design, Setting, and Participants: A case series was conducted from June 29, 1989, to November 12, 2009, of 13 children with PNs arising in CMN in childhood and 5 children with melanomas arising in CMN in childhood. Five patients with giant CMN with no nodules were included as negative controls, and 6 patients with melanomas arising in CMN in adulthood were included as positive controls. Follow-up ranged from 3 to 21 years in all children (mean, 9.9 years) and from 3 months to 7 years in adults. Specimens were selected for immunohistochemistry and FISH. All histopathologic sections were reviewed by 2 dermatopathologists who examined all nodules arising at different ages in the same patient and, in the case of melanoma, all locations. Data analysis was performed from January 1, 2013, to January 31, 2015., Main Outcomes and Measures: The ability to distinguish melanoma from PN using immunohistochemistry and/or FISH., Results: Of the 13 patients (5 boys and 8 girls) with PNs present at birth, all PNs were stable (mean follow-up, 9 years). Eight patients with PNs and 4 of 5 patients with childhood-onset melanoma showed homogeneous staining for HMB45, while heterogeneous staining for HMB45 was seen in 3 of 6 patients with adult-onset melanoma. Expression of p16 was strongly positive in most patients with childhood-onset PNs (10 of 11 patients) and melanoma (all patients) but negative in 4 patients with adult-onset melanoma. Patients with PNs and the 5 patients with childhood-onset melanoma had numerical chromosomal aberrations never observed in the adjacent CMN. The 2 children with FISH-positive PNs are melanoma free after 7 and 4 years. Only 1 patient with childhood-onset melanoma had a FISH aberration compared with 4 patients with adult-onset melanoma., Conclusions and Relevance: Immunohistochemistry and the 4-probe FISH melanoma analysis are not useful for distinguishing PN from childhood-onset melanoma as opposed to adult-onset melanoma. Numerical anomalies seen in PNs but not in the adjacent CMN could be the result of a chromosomal segregation malfunction resulting in the development of nodules.

Purpose: Development of liver metastasis remains the most common cause of mortality in uveal melanoma (UM). A few cell lines cultured from primary UM tumors have been used widely to investigate the pathobiology of UM. However, the translation of basic knowledge to the clinic for the treatment of the metastatic disease has remained incremental at best. In this study, we examined whether the properties of UM cell lines at various passages were similar to their corresponding primary tumors., Methods: Gene expression profiling by microarray was performed on UM primary tumors and derived cell lines cultured at varying passages. Expression of UM protein markers was monitored by immunohistochemical analyses and Western blotting. The in vivo tumorigenic properties of UM cultures were evaluated using athymic nude mice., Results: Cell passaging severely reduced the expression of genes encoding markers typical of UM, including those of the prognostic gene signature. Marked differences between gene expression profiles of primary tumors and cell lines could be linked to the infiltrating immune and stromal cells in situ. In addition, the tumorigenic properties of UM cell lines also increased with cell passaging in culture as evaluated by their subcutaneous injection into athymic mice., Conclusions: Together, these findings demonstrate that the short-term UM primary cultures exhibit molecular features that resemble the respective surgical material and, thus, represent the best model for in vitro-assessed cancer treatments.

As knowledge continues to develop, regular updates are necessary concerning recommendations for practice. The recommendations for the management of melanoma stages I to III were drawn up in 2005. At the request of the Société Française de Dermatologie, they have now been updated using the methodology for recommendations proposed by the Haute Autorité de Santé. In practice, the principal recommendations are as follows: for staging, it is recommended that the 7th edition of AJCC be used. The maximum excision margins have been reduced to 2cm. Regarding adjuvant therapy, the place of interferon has been reduced and no validated emerging medication has yet been identified. Radiotherapy may be considered for patients in stage III at high risk of relapse. The sentinel lymph node technique remains an option. Initial examination includes routine ultrasound as of stage II, with other examinations being optional in stages IIC and III. A shorter strict follow-up period (3years) is recommended for patients, but with greater emphasis on imaging., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Large congenital melanocytic naevi (LCMN) represent the main risk factor for development of melanoma in childhood. This retrospective study of 10 cases of melanoma in patients with LCMN used fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) (6 cases) to elucidate the clinical, histological, and cytogenetic characteristics of this rare disorder. Six melanomas were found within the LCMN, the others in lymph nodes, subcutis and brain. The LCMN was located on the trunk in 8 cases, with satellite naevi in 6 cases. Two distinct groups emerged: 5 melanomas that developed before the age of 10 years and the other after 20 years. The mortality rate was 60% and clearly correlated with clinical stage at diagnosis. Histological diagnosis was difficult in only 2 patients in whom neither immunohistochemistry nor FISH were helpful. Otherwise, CGH showed a high number of chromosomal aberrations leading to a formal diagnosis.

Cutaneous melanoma is a multifactorial disease resulting from both environmental and genetic factors. Five susceptibility genes have been identified over the past years, comprising high-risk susceptibility genes (CDKN2A, CDK4, and BAP1 genes) and intermediate-risk susceptibility genes (MITF, and MC1R genes). The aim of this expert consensus was to define clinical contexts justifying genetic analyses, to describe the conduct of these analyses, and to propose surveillance recommendations. Given the regulatory constraints, it is recommended that dermatologists work in tandem with a geneticist. Genetic analysis may be prescribed when at least two episodes of histologically proven invasive cutaneous melanoma have been diagnosed before the age of 75 years in two 1st or 2nd degree relatives or in the same individual. The occurrence in the same individual or in a relative of invasive cutaneous melanoma with ocular melanoma, pancreatic cancer, renal cancer, mesothelioma or a central nervous system tumour are also indications for genetic testing. Management is based upon properly managed photoprotection and dermatological monitoring according to genetic status. Finally, depending on the mutated gene and the familial history, associated tumour risks require specific management (e.g. ocular melanoma, pancreatic cancer). Due to the rapid progress in genetics, these recommendations will need to be updated regularly., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Trichoblastoma is a benign cutaneous adnexal tumor, composed mostly of follicular germinative cells. Its pigmented variant is colonized by numerous dendritic melanocytes. So far, only one case in the literature describes a combination of trichoblastoma and melanoma. We report the case of a 62-year-old man who had a slow-growing mass of the left flank present since childhood. This 8-cm mass was surgically removed when it became ulcerated and associated with axillary lymph nodes. Histologically, this tumor was strictly dermal and composed of 2 intermingled components. Large sheets of atypical, proliferating epithelioid cells predominated. Dispersed solid nests or cribriform epithelial islets encased in fibrous tissue were also seen. Some nests displayed a massive colonization by pigmented dendritic melanocytes. On immunohistochemical staining, the sheets of atypical cells expressed focally but strongly S100 protein, MelanA, HMB45, and MiTF. Epithelial structures diffusely expressed pancytokeratin AE1/AE3, KL1, and pleckstrin homology-like domain, family A, member 1. Based on these results, we diagnosed an intradermal melanoma, possibly developed from dendritic melanocytes colonizing a giant pigmented trichoblastoma. Direct sequencing of the melanoma revealed a rarely described NRAS mutation c.34G>T (G12C). Array comparative genomic hybridization displayed a complex profile somewhat divergent from standard melanoma profiles. The patient died of widespread metastatic disease 8 months after initial diagnosis.

Imatinib mesylate is used in targeted therapy of cancer to inhibit type III tyrosine kinase receptors, such as KIT and platelet-derived growth factor receptors (PDGFRs). Expression of KIT in uveal melanoma (UM) suggests that this receptor may be the target of imatinib mesylate therapy. However, phase II multicenter clinical studies have shown no effect of imatinib mesylate in patients with unresectable liver metastases of UM. We therefore investigated which molecular mechanisms promote imatinib mesylate-resistance in metastatic UM. Expression of KIT, stem cell factor (SCF), PDGFRα and PDGFRβ, was analyzed by RT-PCR, immunostaining, and Western blot in twenty-four samples of UM liver metastases, as well as UM primary tumor and metastatic cell lines. Soluble SCF was quantified in UM cell lines using enzyme-linked immunosorbent assay. Cell viability of UM cell lines treated with imatinib mesylate and grown in SCF-supplemented medium or in microvascular endothelial cells-conditioned medium was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays. UM liver metastases and cell lines expressed KIT and SCF, but not the PDGFRs. Ninety-five percent of liver metastases expressed KIT at the protein level, but PDGFRs were not detected in these samples. Imatinib mesylate reduced the viability of UM metastatic cell lines in a concentration-dependent manner, but an increased resistance to this drug was observed when cells were incubated in SCF-supplemented or microvascular endothelial cells-conditioned medium. This study provides evidence that tumor microenvironment cytokines such as SCF may promote resistance to imatinib mesylate in metastatic UM.

The outcome of patients presenting with vaginal melanoma has been assessed in a large multicentric retrospective study. The databases of 12 French institutions were searched for primary vaginal melanomas managed between 1990 and 2007. Among the 54 patients recorded, 46 were managed with a curative intent and included in the study. The clinical characteristics, treatments, and detection of c-KIT protein expression have been studied. The median age of the patients was 63.5 years (42-88). Twenty-eight patients were classified as International Federation of Gynecology and Obstetrics (FIGO) stage I, five as stage II, six as stage III, and one as stage IVA. c-KIT protein was overexpressed in 80% of the patients. Forty-two patients underwent surgical resection of the tumor, nine patients received local adjuvant treatment, and 10 received systemic adjuvant therapy. The median relapse-free survival was 10.9 months. c-KIT-negative status (P=0.01) and stage I (P=0.02) were associated with locoregional recurrence. The rate of metastasis was increased for advanced FIGO stages (P<0.01). The median overall survival (OS) was 28.4 months. The finding of lymph node metastasis adversely affected OS (P<0.01). Conservative surgery and radiotherapy were associated with a decrease in metastasis-free and OS (P<0.01) compared with surgery alone, this group of patients presenting with advanced FIGO stages (P=0.02). Despite the use of limited data, conservative surgery combined with a sentinel lymph node procedure, followed by adjuvant radiotherapy could be proposed to patients with early FIGO stage in the absence of validated management. c-KIT negativity by immunochemistry appears to be a poor prognosis marker in terms of locoregional recurrences but not for metastatic spread nor survival. Further assessment of the role of c-KIT expression in this disease is thus mandatory to select patients for targeted therapy., (© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.)

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.

Some melanocytic tumors are ambiguous, so the reproducible histopathological diagnosis of benign or malignant lesion is difficult. This study evaluated the contribution of fluorescence in situ hybridization (FISH) first in 43 non-equivocal melanomas and nevi, and then in 113 ambiguous melanocytic tumors selected by expert pathologists from six different European institutions. We included two groups of ambiguous tumors: patients without recurrence (5-year minimal follow-up) and with metastases. An independent triple-blind histopathological review was performed to classify tumors as 'favor benign' (A-) or 'favor malignant' (A+). A four-color probe set targeting 6p25, 6q23, 11q13 and CEP6 was used for FISH. In the 43 non-equivocal melanomas and nevi, sensitivity was 85% and specificity 90%. Ninety out of 95 ambiguous melanocytic tumors included were FISH interpretable (67 FISH negative and 23 FISH positive). Of the 90 patients, 69 presented no recurrence and 21/90 exhibited metastases. These ambiguous tumors were mostly spitzoid tumors (45/90). Histopathological reviewers classified these tumors as favor malignant (49/90) and favor benign (32/90), whereas nine cases had a discordant diagnosis. By comparison with outcome, the sensitivity and specificity of histopathological review were 95 and 52%, and the sensitivity and specificity of FISH were 43 and 80%. Compared with histopathological review, the sensitivity and specificity of FISH were 34.5 and 91%. Interestingly, by combining the histopathological diagnosis with FISH results, the diagnosis was optimized, especially by increasing specificity (76% instead of 52% for expert diagnosis alone) and by improving sensitivity compared with FISH alone (90 vs 43% for FISH result alone). The value of this FISH test is to add a reproducible demonstration of malignancy to the histopathological diagnosis, especially in doubtful/ambiguous melanocytic tumors. A positive FISH test reinforces the diagnosis of melanoma, allowing such tumors (particularly thick tumors) to be managed as melanomas.

Background: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined., Results and Methods: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal-epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT-PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival., Conclusion: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.

Cutaneous melanomas are exceptional in children and represent a variety of clinical situations, each with a different prognosis. In congenital nevi, the risk of transformation is correlated with the size of the nevus. The most frequent type is lateral transformation, extremely rare before puberty, reminiscent of a superficial spreading melanoma (SSM) ex-nevus. Deep nodular transformation is much rarer, can occur before puberty, and must be distinguished from benign proliferative nodules. Superficial spreading melanoma can also arise within small nevi, which were not visible at birth, usually after puberty, and can reveal a cancer predisposition syndrome (CDKN2A or CDK4 germline mutations). Prognosis is correlated with classical histoprognostic features (mainly Breslow thickness). Spitz tumors are frequent in adolescents and encompass benign (Spitz nevus), intermediate (atypical Spitz tumor), and malignant forms (malignant Spitz tumor). The whole spectrum is characterized by specific morphology with spindled and epithelioid cells, genetic features, and an overall favorable outcome even if a regional lymph node is involved. Nevoid melanomas are rare and difficult to diagnose clinically and histologically. They can arise in late adolescence. Their prognosis is currently not very well ascertained. A small group of melanomas remains unclassified after histological and molecular assessment.

Deep penetrating nevus (DPN) is characterized by enlarged, pigmented melanocytes that extend through the dermis. DPN can be difficult to distinguish from melanoma but rarely displays aggressive biological behavior. Here, we identify a combination of mutations of the β-catenin and mitogen-activated protein kinase pathways as characteristic of DPN. Mutations of the β-catenin pathway change the phenotype of a common nevus with BRAF mutation into that of DPN, with increased pigmentation, cell volume and nuclear cyclin D1 levels. Our results suggest that constitutive β-catenin pathway activation promotes tumorigenesis by overriding dependencies on the microenvironment that constrain proliferation of common nevi. In melanoma that arose from DPN we find additional oncogenic alterations. We identify DPN as an intermediate stage in the step-wise progression from nevus to melanoma. In summary, we delineate specific genetic alterations and their sequential order, information that can assist in the diagnostic classification and grading of these distinctive neoplasms.Deep penetrating nevi (DPN) are unusual melanocytic neoplasms with unknown genetic drivers. Here the authors show that majority of DPN harbor activating mutations in the β-catenin and the MAP-kinase pathways; this characteristic can help in the classification and grading of these distinctive neoplasms.

Abstract Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF‐mutant melanoma. In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching. The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas. Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAFV600‐mutated cell lines and tumors. ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment. ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITFlow/p75high stem‐like and tumorigenic phenotype. ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells. Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi.