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Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a ∼50% response rate to immune checkpoint blockade (ICB) therapy. To identify predictive biomarkers, we integrated bulk and single-cell RNA sequencing (RNA-seq) with spatial transcriptomics from a cohort of 186 samples from 116 patients, including bulk RNA-seq from 14 matched pairs pre- and post-ICB. In nonresponders, tumors show evidence of increased tumor proliferation, neuronal stem cell markers, and IL1. Responders have increased type I/II interferons and preexisting tissue resident (Trm) CD8 or Vδ1 γδ T cells that functionally converge with overlapping antigen-specific transcriptional programs and clonal expansion of public T-cell receptors. Spatial transcriptomics demonstrated colocalization of T cells with B and dendritic cells, which supply chemokines and costimulation. Lastly, ICB significantly increased clonal expansion or recruitment of Trm and Vδ1 cells in tumors specifically in responders, underscoring their therapeutic importance. These data identify potential clinically actionable biomarkers and therapeutic targets for MCC. Significance: MCC serves as a model of ICB response. We utilized the largest-to-date, multimodal MCC dataset (n = 116 patients) to uncover unique tumor-intrinsic properties and immune circuits that predict response. We identified CD8 Trm and Vδ1 T cells as clinically actionable mediators of ICB response in major histocompatibility complex-high and -low MCCs, respectively., (©2024 American Association for Cancer Research.)

Merkel cell polyomavirus (MCPyV) is a significant contributor to the development of Merkel cell carcinoma (MCC), an aggressive skin cancer with high recurrence and a low survival rate. In fact, it is the deadliest skin cancer. The precise routes of transmission for MCPyV-positive MCC remain unclear, but several factors may trigger its development. Conventional treatments for MCC are not highly effective, especially in patients with metastasis, with a clear need for new treatment options. Gene-targeted therapies hold great promise for the treatment of MCC, including the use of siRNA and CRISPR/Cas (C/Cas) but critically none have yet been translated into clinical trials. Validating this approach is the fact that several siRNA products are already FDA licenced, while C/Cas has entered clinical trial, albeit for conditions other than MCC. There are many challenges that must be overcome to move from preclinical research to the clinic. In this review, we provide a comprehensive summary of the current understanding of MCC, with a particular focus on MCPyV-positive MCC, and the status of gene-targeted therapies. Additionally, we discuss the major obstacles that impede MCC research and explore future prospects., (© 2024 The Author(s). Reviews in Medical Virology published by John Wiley & Sons Ltd.)

Competing Interests: The authors declare no conflicts of interest.

Cutaneous squamous cell carcinoma (cSCC) is a neoplasm type often diagnosed in dogs. However, studies focused on further investigating its molecular biology, mainly biomarkers to help implementing new therapies, remain scare in the literature. Thus, immunostaining and the gene expression of epidermal growth factor receptors (HER1 and HER2) in canine cSCC presenting different cell differentiation degrees were herein assessed. Thirty-two (32) canine cSCC were selected, classified based on to their cell differentiation degree and subjected to immunohistochemical study to assess HER1 and HER2 immunostaining intensity and distribution. In addition, HER1 and HER2 gene expression was investigated through real-time PCR. Membranous and cytoplasmic immunostaining were observed in both markers. HER2 prevailed in poorly differentiated cSCC; there was positive protein expression correlation between both markers. Mean HER1 gene expression was higher in moderately differentiated, whereas mean HER2 gene expression was higher in poorly differentiated cSCC. Moreover, there was gene expression correlation between markers, regardless of cell differentiation degree. Thus, HER2 protein immunostaining and gene expression were higher in poorly differentiated canine cSCC and it enabled understanding that increase observed in this epidermal growth factor receptor is proportional to this neoplasm's cell differentiation degree in canine species. Results in the current study helped better understanding canine cSCC's molecular biology; however, it is relevant studying other markers aiming to investigate signaling pathways., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by genomic integration of the Merkel cell polyomavirus (MCPyV). MCPyV-negative cases often present as combined MCCs, which represent a distinctive subset of tumors characterized by association of an MCC with a second tumor component, mostly squamous cell carcinoma. Up to now, only exceptional cases of combined MCC with neuroblastic differentiation have been reported. Herein we describe two additional combined MCCs with neuroblastic differentiation and provide comprehensive morphologic, immunohistochemical, transcriptomic, genetic and epigenetic characterization of these tumors, which both arose in elderly men and appeared as an isolated inguinal adenopathy. Microscopic examination revealed biphasic tumors combining a poorly differentiated high-grade carcinoma with a poorly differentiated neuroblastic component lacking signs of proliferation. Immunohistochemical investigation revealed keratin 20 and MCPyV T antigen (TA) in the MCC parts, while neuroblastic differentiation was confirmed in the other component in both cases. A clonal relation of the two components can be deduced from 20 and 14 shared acquired point mutations detected by whole exome analysis in both combined tumors, respectively. Spatial transcriptomics demonstrated a lower expression of stem cell marker genes such as SOX2 and MCM2 in the neuroblastic component. Interestingly, although the neuroblastic part lacked TA expression, the same genomic MCPyV integration and the same large T-truncating mutations were observed in both tumor parts. Given that neuronal transdifferentiation upon TA repression has been reported for MCC cell lines, the most likely scenario for the two combined MCC/neuroblastic tumors is that neuroblastic transdifferentiation resulted from loss of TA expression in a subset of MCC cells. Indeed, DNA methylation profiling suggests an MCC-typical cellular origin for the combined MCC/neuroblastomas. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Melanomas lacking mutations in BRAF, NRAS and NF1 are frequently referred to as "triple wild-type" (tWT) melanomas. They constitute 5-10 % of all melanomas and remain poorly characterized regarding clinical characteristics and response to therapy. This study investigates the largest multicenter collection of tWT-melanomas to date., Methods: Targeted next-generation sequencing of the TERT promoter and 29 melanoma-associated genes were performed on 3109 melanoma tissue samples of the prospective multicenter study ADOREG/TRIM of the DeCOG revealing 292 patients suffering from tWT-melanomas. Clinical characteristics and mutational patterns were analyzed. As subgroup analysis, we analyzed 141 tWT-melanoma patients receiving either anti-CTLA4 plus anti-PD1 or anti PD1 monotherapy as first line therapy in AJCC stage IV., Results: 184 patients with cutaneous melanomas, 56 patients with mucosal melanomas, 34 patients with acral melanomas and 18 patients with melanomas of unknown origin (MUP) were included. A TERT promoter mutation could be identified in 33.2 % of all melanomas and 70.5 % of all tWT-melanomas harbored less than three mutations per sample. For the 141 patients with stage IV disease, mPFS independent of melanoma type was 6.2 months (95 % CI: 4-9) and mOS was 24.8 months (95 % CI: 14.2-53.4) after first line anti-CTLA4 plus anti-PD1 therapy. After first-line anti-PD1 monotherapy, mPFS was 4 months (95 %CI: 2.9-8.5) and mOS was 29.18 months (95 % CI: 17.5-46.2)., Conclusions: While known prognostic factors such as TERT promoter mutations and TMB were equally distributed among patients who received either anti-CTLA4 plus anti-PD1 combination therapy or anti-PD1 monotherapy as first line therapy, we did not find a prolonged mPFS or mOS in either of those. For both therapy concepts, mPFS and mOS were considerably shorter than reported for melanomas with known oncogene mutations., Competing Interests: Declaration of Competing Interest All other authors declare no conflicts of interest for the submitted work., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sarcoids are the most frequently diagnosed dermatological tumour in horses. It is a disease that can affect various species of equids, such as donkeys, mules and zebras. This type of tumour can develop in all horse breeds, regardless of age and gender. Treatment options depend on many factors, such as the type of lesion, location, extent, owner preference and financial considerations. In the present study, we investigated the TRIM29 expression, the methylation status of its first exon and its involvement in the formation of equine sarcoids. Bisulfite sequencing PCR (BSP) was used to determine DNA methylation at CpG sites and real-time quantitative polymerase chain reaction (qPCR) was used to detect TRIM29 expression level. Our results showed that TRIM29 is significantly downregulated in lesional samples (FC = -3.72; p < 0.001). Furthermore, TRIM29 expression was significantly correlated (R = -0.73; p < 0.001) with hypermethylation of its specific CpG sites in the first exon of this gene. Our research has demonstrated that the identification of increased methylation of CpG sequences in horse sarcoids, along with the decreased expression of the TRIM29 gene, is an important step towards understanding the molecular mechanisms underlying the disease. These findings can serve in the future as a diagnostic biomarker for horse sarcoids and help in detecting the disease., (© 2024 John Wiley & Sons Ltd.)

Melanoma pathogenesis, conventionally perceived as a linear accumulation of molecular changes, discloses substantial heterogeneity driven by non-linear biological processes, including the direct transformation of melanocyte stem cells. This heterogeneity manifests in diverse biological phenotypes and developmental states, influencing variable responses to treatments. Unveiling the aberrant mechanisms steering melanoma initiation, progression, and metastasis is imperative. Beyond mutations in oncogenic and tumor suppressor genes, the involvement of distinct molecular pathways assumes a pivotal role in melanoma pathogenesis. Ultraviolet (UV) radiations, a principal factor in melanoma etiology, categorizes melanomas based on cumulative sun damage (CSD). The genomic landscape of lesions correlates with UV exposure, impacting mutational load and spectrum of mutations. The World Health Organization's 2018 classification underscores the interplay between sun exposure and genomic characteristics, distinguishing melanomas associated with CSD from those unrelated to CSD. The classification elucidates molecular features such as tumor mutational burden and copy number alterations associated with different melanoma subtypes. The significance of the mutated BRAF gene and its pathway, notably BRAFV600 variants, in melanoma is paramount. BRAF mutations, prevalent across diverse cancer types, present therapeutic avenues, with clinical trials validating the efficacy of targeted therapies and immunotherapy. Additional driver mutations in oncogenes further characterize specific melanoma pathways, impacting tumor behavior. While histopathological examination remains pivotal, challenges persist in molecularly classifying melanocytic tumors. In this review, we went through all molecular characterization that aid in discriminating common and ambiguous lesions. Integration of highly sensitive molecular diagnostic tests into the diagnostic workflow becomes indispensable, particularly in instances where histology alone fails to achieve a conclusive diagnosis. A diagnostic algorithm based on different molecular features inferred by the various studies is here proposed., Competing Interests: Declaration of Competing Interest Authors declare no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Melanoma metabolism can be reprogrammed by activating BRAF mutations. These mutations are present in up to 50% of cutaneous melanomas, with the most common being V600E. BRAF mutations augment glycolysis to promote macromolecular synthesis and proliferation. Prior to the development of targeted anti-BRAF therapies, these mutations were associated with accelerated clinical disease in the metastatic setting. Combination BRAF and MEK inhibition is a first line treatment option for locally advanced or metastatic melanoma harboring targetable BRAF mutations. This therapy shows excellent response rates but these responses are not durable, with almost all patients developing resistance. When BRAF mutated melanoma cells are inhibited with targeted therapies the metabolism of those cells also changes. These cells rely less on glycolysis for energy production, and instead shift to a mitochondrial phenotype with upregulated TCA cycle activity and oxidative phosphorylation. An increased dependence on glutamine utilization is exhibited to support TCA cycle substrates in this metabolic rewiring of BRAF mutated melanoma. Herein we describe the relevant core metabolic pathways modulated by BRAF inhibition. These adaptive pathways represent vulnerabilities that could be targeted to overcome resistance to BRAF inhibitors. This review evaluates current and future therapeutic strategies that target metabolic reprogramming in melanoma cells, particularly in response to BRAF inhibition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Squamous cell carcinomas (SCCs), including lung, head & neck, bladder, and skin SCCs often display constitutive activation of the KEAP1-NRF2 pathway. Constitutive activation is achieved through multiple mechanisms, including activating mutations in NFE2L2 (NRF2). To determine the functional consequences of Nrf2 activation on skin SCC development, we assessed the effects of mutant Nrf2 E79Q expression, one of the most common activating mutations in human SCCs, on tumor promotion and progression in the mouse skin multistage carcinogenesis model using a DMBA-initiation/TPA-promotion protocol where the Hras A->T mutation (Q61L) is the canonical driver mutation. Nrf2 E79Q expression was temporally and conditionally activated in the epidermis at two stages of tumor development: 1) after DMBA initiation in the epidermis but before cutaneous tumor development and 2) in pre-existing DMBA-initiated/TPA-promoted squamous papillomas. Expression of Nrf2 E79Q in the epidermis after DMBA initiation but before tumor occurrence inhibited the development/promotion of 70% of squamous papillomas. However, the remaining papillomas often displayed non-canonical Hras and Kras mutations and enhanced progression to SCCs compared to control mice expressing wildtype Nrf2. Nrf2 E79Q expression in pre-existing tumors caused rapid regression of 60% of papillomas. The remaining papillomas displayed the expected canonical Hras A->T mutation (Q61L) and enhanced progression to SCCs. These results demonstrate that mutant Nrf2 E79Q enhances the promotion and progression of a subset of skin tumors and alters the frequency and diversity of oncogenic Ras mutations when expressed early after initiation., Competing Interests: Declaration of competing interest All authors have declared they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Most clinical trials investigating targeted therapies for patients harboring BRAF V600 mutations have included mostly White patients, and data for Asian patients are scarce. Although there are several retrospective studies in Japanese patients, they have investigated only the dabrafenib + trametinib regimen, and have had a short follow-up period. We conducted a single-center retrospective study to update previous studies and compare the outcomes with those in White patients. We analyzed 89 patients who received dabrafenib + trametinib or encorafenib + binimetinib, including 11 who received both treatment regimens. The overall response rate was 79.8%, with complete response in 25 patients (28.1%) and partial response in 45 patients (51.7%). The median progression-free survival was 13.7 months, and the median overall survival was 32.9 months. The 3-year progression-free and overall survival rates were 31.8% and 47.9%, respectively. Although the two regimens showed no significant differences in efficacy, their safety profiles differed, as reported in clinical trials. Therefore, the most frequent adverse event associated with the dabrafenib + trametinib regimen was pyrexia (61.3%) and that of encorafenib + binimetinib was blurred vision (32.0%). Switching directly to another targeted therapy after progressive disease showed no clinical response; however, rechallenge followed by immune checkpoint inhibitor therapy showed a certain response. As a prognostic factor, performance status was associated with progression-free survival, and performance status, serum lactate dehydrogenase level, and dose interruption were associated with overall survival in the multivariate analysis. Real-world data on targeted therapy for patients with melanoma in Japan suggest that both dabrafenib + trametinib and encorafenib + binimetinib show similar efficacy and safety in Asian and White patients., (© 2024 Japanese Dermatological Association.)

Acral lentiginous melanoma (ALM) is the most common subtype of acral melanoma. Even though recent genetic studies are reported in acral melanomas, the genetic differences between in-situ and invasive ALM remain unclear. We aimed to analyze specific genetic changes in ALM and compare genetic differences between in-situ and invasive lesions to identify genetic changes associated with the pathogenesis and progression of ALM. We performed whole genome sequencing of 71 tissue samples from 29 patients with ALM. Comparative analyses were performed, pairing in-situ ALMs with normal tissues and, furthermore, invasive ALMs with normal and in-situ tissues. Among 21 patients with in-situ ALMs, 3 patients (14.3%) had SMIM14 , SLC9B1 , FRG1 , FAM205A , ESRRA , and ESPN mutations, and copy number (CN) gains were identified in only 2 patients (9.5%). Comparing 13 invasive ALMs with in-situ tissues, CN gains were identified in GAB2 in 8 patients (61.5%), PAK1 in 6 patients (46.2%), and UCP2 and CCND1 in 5 patients (38.5%). Structural variants were frequent in in-situ and invasive ALM lesions. Both in-situ and invasive ALMs had very low frequencies of common driver mutations. Structural variants were common in both in-situ and invasive ALMs. Invasive ALMs had markedly increased CN gains, such as GAB2 , PAK1 , UCP2 , and CCND1 , compared with in-situ lesions. These results suggest that they are associated with melanoma invasion., 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 © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

In patients with resected non-metastatic melanoma, the liquid biopsy for the assessment of molecular residual disease (MRD) by circulating tumour DNA (ctDNA) represents a promising tool to stratify the risk and to monitor tumour evolution. However, its validation requires the demonstration of analytical validity, clinical validity and utility. Indeed, the development of sensitive and specific assays can optimize prognostication and eventually help clinicians to modulate adjuvant treatments, in order to improve clinical outcomes. Data about ctDNA-guided prognosis stratification is emerging, but clinical trials assessing ctDNA-guided therapeutic decisions are still ongoing. This review aims to depict the role of ctDNA-based MRD assessment in patients with non-metastatic melanoma and to provide a roadmap to face challenges for its introduction into clinical practice., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PQ reports consulting fees and honoraria from Bristol Myers Squibb, Merck, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi, and Sun Pharma, and travel, accommodations, or other expenses from Merck Sharp & Dohme and Sanofi; FS received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Novartis, MSD, BMS, Pierre Fabre, Merck, Sanofi, Sun Pharma, IGEA, Philogen, and received consulting fees from Novartis, MSD, Sun Pharma, Pierre Fabre, Philogen; AO has been principal investigator in clinical trials sponsored by AbbVie, Alfasigma, Almirall, Amgen, Difa Cooper, Celgene, Eli-Lilly, Galderma, Janssen, Laboratori Farmacologici Milanesi, Leo Pharma, Novartis, Pfizer, Pierre Fabre, Regeneron, Sanofi, UCB-Pharma, and Boehringer-Ingelheim. All other authors have no potential conflict of interest to disclose., (Copyright © 2024. Published by Elsevier Ltd.)

Cutaneous spindle cell neoplasms can be challenging to diagnose using routine histopathological techniques alone, and the growing repertoire of molecular studies can assist in diagnosis. We describe a cutaneous spindle cell neoplasm characterized by a COL3A1::PDGFRA rearrangement predicted to lead to constitutive activation of the PDGFRA kinase domain. The lesion shows some similarities to dermatofibrosarcoma protuberans and also benign and epithelioid fibrous histiocytomas but is distinct from these entities histopathologically and molecularly. This tumor is considered to represent an entity in the spectrum of PDGFR-driven cutaneous mesenchymal neoplasms., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

During routine dermatologic examination, a 77-year-old male was noted to have a firm blue subcutaneous nodule on his right lateral upper back. His past medical history included metastatic melanoma of unknown primary involving right and left axillary lymph nodes, treated with ipilimumab/nivolumab with complete response, and subsequent primary uveal melanoma. The subcutaneous nodule was located near his previous right axillary scar for metastatic melanoma. Excision of the nodule showed a plexiform neoplasm involving mid and deep dermis composed of spindle and epithelioid atypical cells admixed with numerous melanophages. Central necrosis was present. Immunohistochemical studies revealed the tumor cells to be diffusely positive for HMB45, with retained expression of BAP1 and p16. The tumor cells were negative for PRAME, nuclear expression of β-catenin, LEF1, and BRAF V600E. Molecular studies demonstrated BAP1 and GNA11 somatic mutations, a profile different from that exhibited by his prior melanoma. Collectively, these data were interpreted as a metastasis from uveal melanoma and not a recurrence of his metastatic likely cutaneous melanoma after complete response to immunotherapy. This case emphasizes the importance of molecular studies for definitive diagnosis in challenging clinical situations, especially when there is discordance among histopathological, immunohistochemical, and molecular studies. Integration of clinical, histopathological, and molecular features is warranted., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Immunotherapy has transformed the treatment of advanced melanoma. However, up to two-thirds of patients experience disease progression after initially achieving a response to immunotherapy. Furthermore, most research has focused on cutaneous melanoma rather than acral or mucosal melanoma, although the latter predominates in Asian populations. In this review, we examine and summarize current definitions of resistance to immunotherapy and the epidemiology of resistance to PD-1 inhibition. We also review the available literature on molecular mechanisms of resistance, including how the tumor mutational landscape and tumor microenvironments of immunotherapy-resistant acral and mucosal melanomas may influence resistance. Finally, we review strategies for overcoming resistance to PD-1 inhibition and summarize completed studies and ongoing clinical trials. Our review highlights that improving the understanding of resistance mechanisms, optimizing existing therapies and further studying high-risk populations would maximize the potential of immunotherapy and result in optimized treatment outcomes for patients with melanoma., Competing Interests: Declaration of Competing Interest Hongwei Fan and Li Zhang are employees of MSD China. The other authors have no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm 2 and 166.7 cells/mm 2 , respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation., (© 2024 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Abstract: PCR-based fragment analysis of the T-cell receptor (TCR) gene is used extensively in diagnostic labs to assess clonality in T-cell populations in multiple tissue sites. Of the numerous TCR assays that have been reported, studies assessing use on biopsies suspicious for mycosis fungoides specifically are lacking. We compared clonality findings from a previously run 2-tube/2-fluorochrome dye assay to a redesigned 1-tube/1-fluorochrome dye assay on formalin-fixed skin biopsies. Overall, the accuracy of the 2-tube assay was marginally better (75.7% vs. 71.4%), when using clinical history combined with histologic diagnosis as the gold standard. The 2-tube assay had better sensitivity (73.7% vs. 65.8%), while the 1-tube assay had superior specificity (93.8% vs. 87.5%). Clonality results were easier to interpret with the 1-tube assay. In nearly 19% of cases, a change of assays on the same biopsy resulted in a change of clonality interpretation. For laboratories that change TCR-γ clonality assays, follow-up biopsies for mycosis fungoides assessment may result in a change of diagnosis., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Cutaneous neurofibromas (CNFs) are benign tumors that occur in the dermis of individuals with the inherited tumor predisposition disorder, neurofibromatosis type 1. CNFs cause disfigurement, pain, burning, and itching, resulting in substantially reduced QOL in patients with neurofibromatosis type 1. CNFs are benign tumors that exhibit cellular and molecular heterogeneity, making it difficult to develop tractable in vitro or in vivo models. As a result, CNF research and drug discovery efforts have been limited. To address this need, we developed a reproducible patient-derived explant (PDE) ex vivo culture model using CNF tumors from patients with neurofibromatosis type 1. CNF PDEs remain viable in culture for over 9 days and recapitulate the cellular composition and molecular signaling of CNFs. Using CNF PDEs as a model system, we found that proliferation was associated with increased T-cell infiltration. Furthermore, we identified a pattern of reciprocal inflammatory signaling in CNF PDEs in which tumors rely on prostaglandin or leukotriene-mediated signaling pathways. As proof of principle, we show that ex vivo glucocorticoid treatment reduced the expression of proinflammatory genes, confirming that CNF PDEs are a useful model for both mechanistic studies and preclinical drug testing., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Genotype-phenotype associations in recessive dystrophic epidermolysis bullosa (RDEB) have been difficult to elucidate., Objective: To investigate RDEB genotype-phenotype associations and explore a functional approach to genotype classification., Methods: Clinical examination and genetic testing of RDEB subjects, including assessment of clinical disease by RDEB subtype and extent of blistering. Genotypes were evaluated according to each variant's effect on type VII collagen function per updated literature and subsequently categorized by degree of impact on VII collagen function as low-impact (splice/missense, missense/missense), medium-impact (premature termination codon [PTC]/missense, splice/splice), and high-impact (PTC/PTC, PTC/splice). Genotype-phenotype associations were investigated using Kruskal-Wallis and Fisher's exact tests, and age-adjusted regressions., Results: Eighty-three participants were included. High-impact variants were associated with worse RDEB subtype and clinical disease, including increased prevalence of generalized blistering (55.6% for low-impact vs 72.7% medium-impact vs 90.4% high-impact variants, P = .002). In age-adjusted regressions, participants with high-impact variants had 40.8-fold greater odds of squamous cell carcinoma compared to low-impact variants (P = .02), and 5.7-fold greater odds of death compared to medium-impact variants (P = .05)., Limitations: Cross-sectional design., Conclusion: Functional genotype categories may stratify RDEB severity; high-impact variants correlated with worse clinical outcomes. Further validation in larger cohorts is needed., Competing Interests: Conflicts of interest Drs Tang and Marinkovich are investigators for clinical trials sponsored by Abeona Therapeutics, Inc, and Phoenix Tissue Repair. Dr Marinkovich is also an investigator for clinical trials sponsored by WINGS, Castle Creek Pharmaceuticals, and Krystal Biotech. Dr Gorell is a consultant for Krystal Biotech. Dr So, Dr Nazaroff, Dr Yenamandra, Author Harris, Dr Fulchand, Dr Eid, and Author Dolorito have no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier Inc.)

Melanoma poses a poor prognosis with high mortality rates upon metastasis. Exploring the molecular mechanisms governing melanoma progression paves the way for developing novel approaches to control melanoma metastasis and ultimately enhance patient survival rates. Extracellular galectin-3 (Gal-3) has emerged as a pleiotropic promoter of melanoma metastasis, exerting varying activities depending on its interacting partner. However, whether intracellular Gal-3 promotes melanoma aggressive behavior remains unknown. In this study, we explored Gal-3 expression in human melanoma tissues as well as in murine melanoma models to examine its causal role in metastatic behavior. We found that Gal-3 expression is downregulated in metastatic melanoma tissues compared with its levels in primary melanomas. Enforced silencing of Gal-3 in melanoma cells promoted migration, invasion, colony formation, in vivo xenograft growth, and metastasis and activated canonical oncogenic signaling pathways. Moreover, loss of Gal-3 in melanoma cells resulted in upregulated the expression of the prometastatic transcription factor NFAT1 and its downstream metastasis-associated proteins, matrix metalloproteinase 3, and IL-8. Overall, our findings implicate melanoma intracellular Gal-3 as a major determinant of its metastatic behavior and reveal a negative regulatory role for Gal-3 on the expression of NFAT1 in melanoma cells., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated ras Ha /fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTEN flx/flx ). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2 166 activation and sporadic p-AKT 473 expression. In bi-genic HK1.fos/Δ5PTEN flx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTEN flx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2 166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTEN flx/flx papillomas exhibited increasing basal-layer p-MDM2 166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1 473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1 473 . Analysis of TPA-promoted HK1.ras-Δ5PTEN flx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2 166 /p-AKT1 473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1 ras61 papilloma keratinocytes was unexpectedly detected in malignant T52 ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression., (© 2024 The Author(s). Molecular Carcinogenesis published by Wiley Periodicals LLC.)

The barrier function of skin epidermis is crucial for our bodies to interface with the environment. Because epidermis continuously turns over throughout the lifetime, this barrier must be actively maintained by regeneration. Although several transcription factors have been established as essential activators in epidermal differentiation, it is unclear whether additional factors remain to be identified. In this study, we show that CASZ1, a multi zinc-finger transcription factor previously characterized in nonepithelial cell types, shows highest expression in skin epidermis. CASZ1 expression is upregulated during epidermal terminal differentiation. In addition, CASZ1 expression is impaired in several skin disorders with impaired barrier function, such as atopic dermatitis, psoriasis, and squamous cell carcinoma. Using transcriptome profiling coupled with RNA interference, we identified 674 differentially expressed genes with CASZ1 knockdown. Downregulated genes account for 91.2% of these differentially expressed genes and were enriched for barrier function. In organotypic epidermal regeneration, CASZ1 knockdown promoted proliferation and strongly impaired multiple terminal differentiation markers. Mechanistically, we found that CASZ1 upregulation in differentiation requires the action of both the master transcription factor, p63, and the histone acetyltransferase, p300. Taken together, our findings identify CASZ1 as an essential activator of epidermal differentiation, paving the way for future studies understanding of CASZ1 roles in skin disease., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Our goal was to compare molecular and immune profiles of vulvovaginal melanoma (VVM) with cutaneous melanoma (CM) and explore the significance of immune checkpoint inhibitor (ICI) agents on survival., Methods: Samples from VVM and CM tumors underwent comprehensive molecular and immune profiling. Treatment and survival data were extracted from insurance claims data and OS was calculated from time of ICI treatment to last contact. Statistical significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons., Results: Molecular analysis was performed on 142 VVM and 3823 CM tumors. VVM demonstrated significantly (q < 0·01) less frequent BRAF and more frequent KIT, ATRX, and SF3B1 mutations. Alterations in pathways involving DNA damage and mRNA splicing were more common in VVM, while alterations in cell cycle and chromatin remodeling were less common. Immunogenicity of VVM was lower than CM, with an absence of high TMB (0% vs 46.9%) and lower PD-L1 positivity (18·0% vs 29·5%). Median immune checkpoint gene expression was lower in VVM, as were cell fractions for type I macrophages and CD8+ T-cells(q < 0·01). Myeloid dendritic cells were increased in VVM(q < 0·01). Median OS was shorter for VVM than for CM patients treated with ICIs (17·6 versus 37·9 months, HR:1·65 (95% CI 1·02-2·67) p = 0·04)., Conclusions: VVM has a distinct molecular and immune profile compared to CM, which may contribute to the worse survival in VVM compared to CM patients treated with ICI therapy. Though ICIs have been a mainstay of treatment in recent years, our findings suggest that new therapeutic strategies are needed., Competing Interests: Declaration of competing interest SW and JX are employees of Caris Life Sciences. GTG reports grants from Exelixis and Luceno Dynamics; fees for consulting for Bristol Myers Squibb, Merck, Novartis, Regeneron, Immunocore, Iovance, Lyell Immunopharma, Eisai, Incyte, Pfizer, Sapience Therapeutics, Exicure, and Genentech; fees for payment/honoraria from Immunocore; fees for participation on a Data Safety Monitoring Board/Advisory Board for Huyabio. GKI reports fees for consulting for Sanofi and Pfizer; fees for payment/honoraria from Merck, Sanofi, and Regeneron; fees for support for attending meetings from BMS and Sanofi; fees for participation on a Data Safety Monitoring Board/Advisory Board from Replimune, Castle, and Regeneron; institutional research support from BMS, Regeneron, Replimune, Pfizer, InstilBio, Checkmate Pharmaceuticals, and Xencor. TJH reports consulting fees from J & J, Clovis, AstraZeneca, GSK, Roche Genentech, Caris, Genelux, Aravive, Merck, Eisai, and Seagen. BKE reports fees for participation on a Data Safety Monitoring Board/Advisory Board for Merck and GSK. JB reports fees for consulting for Caris, AstraZeneca, Genentech, GSK Tesaro, and Verastem; fees for participation on a Data Safety Monitoring Board/Advisory Board from Caris, AstraZeneca, GSK Tesaro, and Verastem; fees for a leadership/fiduciary role from American Association of Gynecologic Laparoscopy, and Society of Gynecologic Oncology. The remaining authors have nothing to disclose. All of these disclosures mentioned above are unrelated to this study. No author declares a conflict of interest related to the study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Up to 88% of sentinel lymph node biopsies (SLNBs) are negative. The 31-gene expression profile (31-GEP) test can help identify patients with a low risk of SLN metastasis who can safely forego SLNB. The 31-GEP classifies patients as low (Class 1 A), intermediate (Class 1B/2A), or high risk (Class 2B) for recurrence, metastasis, and SLN positivity. The integrated 31-GEP (i31-GEP) combines the 31-GEP risk score with clinicopathologic features using a neural network algorithm to personalize SLN risk prediction., Methods: Patients from a single surgical center with 31-GEP results were included (n = 156). An i31-GEP risk prediction < 5% was considered low risk of SLN positivity. Chi-square was used to compare SLN positivity rates between groups., Results: Patients considered low risk by the i31-GEP had a 0% (0/30) SLN positivity rate compared to a 31.9% (30/94, p < 0.001) positivity rate in those with > 10% risk. Using the i31-GEP to guide SLNB decisions could have significantly reduced the number of unnecessary SLNBs by 19.2% (30/156, p < 0.001) for all patients and 33.0% (30/91, p < 0.001) for T1-T2 tumors. Patients with T1-T2 tumors and an i31-GEP-predicted SLN positivity risk > 10% had a similar SLN positivity rate (33.3%) as patients with T3-T4 tumors (31.3%)., Conclusion: The i31-GEP identified patients with < 5% risk of SLN positivity who could safely forego SLNB. Combining the 31-GEP with clinicopathologic features for a precise risk estimate can help guide risk-aligned patient care decisions for SLNB to reduce the number of unnecessary SLNBs and increase the SLNB positivity yield if the procedure is performed., (© 2024. The Author(s).)

Melanoma, arising from the malignant transformation of melanocytes, stands as the most lethal type of skin cancer. While significant strides have been made in targeted therapy and immunotherapy, substantially enhancing therapeutic efficacy, the prognosis for melanoma patients remains unoptimistic. SIRT7, a nuclear-localized deacetylase, plays a pivotal role in maintaining cellular homeostasis and adapting to external stressors in melanoma, with its activity closely tied to intracellular nicotinamide adenine dinucleotide (NAD + ). However, its involvement in adaptive resistance to targeted therapy remains unclear. Herein, we unveil that up-regulated SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma. Initially, we observed a significant increase of SIRT7 expression in publicly available datasets following targeted therapy within a short duration. In consistent, we found elevated SIRT7 expression in melanoma cells subjected to BRAF or MEK inhibitors in vitro. The up-regulation of SIRT7 expression was also confirmed in xenograft tumors in mice after targeted therapy in vivo. Furthermore, we proved that SIRT7 deficiency led to decreased cell viability upon prolonged exposure to BRAF or MEK inhibitors, accompanied by an increase in cell apoptosis. Mechanistically, SIRT7 deficiency restrained the upregulation of genes associated with mitochondrial biogenesis and intracellular ATP levels in response to targeted therapy treatment in melanoma cells. Ultimately, we proved that SIRT7 deficieny could sensitize BRAF-mutant melanoma cells to MAPK inhibition targeted therapy in vivo. In conclusion, our findings underscore the role of SIRT7 in fostering adaptive resistance to targeted therapy through the facilitation of mitochondrial biogenesis. Targeting SIRT7 emerges as a promising strategy to overcome MAPK inhibitor adaptive resistance in melanoma., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: New diagnostic criteria for NF2-related schwannomatosis (NF2) were published in 2022. An updated UK prevalence was generated in accordance with these, with an emphasis on the rate of de novo NF2 (a 50% frequency is widely quoted in genetic counselling). The distribution of variant types among de novo and familial NF2 cases was also assessed., Methods: The UK National NF2 database identifies patients meeting updated NF2 criteria from a highly ascertained population cared for by England's specialised service. Diagnostic prevalence was assessed on 1 February 2023. Molecular analysis of blood and, where possible, tumour specimens for NF2, LZTR1 and SMARCB1 was performed., Results: 1084 living NF2 patients were identified on prevalence day (equivalent to 1 in 61 332). The proportion with NF2 inherited from an affected parent was only 23% in England. If people without a confirmed molecular diagnosis or bilateral vestibular schwannoma are excluded, the frequency of de novo NF2 remains high (72%). Of the identified de novo cases, almost half were mosaic. The most common variant type was nonsense variants, accounting for 173/697 (24.8%) of people with an established variant, but only 18/235 (7.7%) with an inherited NF2 pathogenic variant (p<0.0001). Missense variants had the highest proportion of familial association (56%). The prevalence of LZTR1 -related schwannomatosis and SMARCB1 -related schwannomatosis was 1 in 527 000 and 1 in 1.1M, respectively, 8.4-18.4 times lower than NF2., Conclusions: This work confirms a much higher rate of de novo NF2 than previously reported and highlights the benefits of maintaining patient databases for accurate counselling., Competing Interests: Competing interests: DGE has received consultancy fees from AstraZeneca, Springworks and Everything Genetic Ltd., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

POT1 is the second most frequently reported gene (after CDKN2A ) in familial melanoma. Pathogenic variants are associated with earlier onset and/or multiple primary melanomas (MPMs). To date, POT1 phenotypical reports have been largely restricted to associated malignancies, and description of the dermatological landscape has been limited. We identified 10 variants in n=18 of 384 (4.7%) unrelated individuals (n=13 MPMs; n=5 single primary melanomas) of European ancestry. Five variants were rare (minor allele frequency <0.001) or novel (two loss-of-function (LOF), one splice acceptor and two missense) and were predicted to be functionally significant, in five unrelated probands with MPMs (≥3 melanomas). We performed three-dimensional total body photography on both individuals with confirmed pathogenic LOF variants to characterise the dermatological phenotype. Total body naevus counts (≥2 mm diameter) were significantly higher (p=7.72 ×10-12 ) in carriers compared with a control population. Majority of naevi were on the probands' back and lower limb regions, where only mild to moderate ultraviolet (UV) damage was observed. Conversely, the head/neck region, where both probands exhibited severe UV damage, had comparably fewer naevi. We hypothesise that carriage of functionally significant POT1 variants is associated with increased naevus counts generally, and naevi >5 mm in diameter specifically and the location of these are independent of UV damage., Competing Interests: Competing interests: PS is a shareholder of MoleMap Limited and e-derm consult and undertakes regular teledermatological reporting for both companies. PS is a medical consultant for Canfield Scientific and Blaze Bioscience MoleMap Australia Limited, and a medical advisor for First Derm. No other authors have conflicts to declare., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

An optimum safety excision margin (EM) delineated by precise demarcation of field cancerization along with reliable biomarkers that enable predicting and timely evaluating patients' response to immunotherapy significantly impact effective management of melanoma. In this study, optimized biphasic "immunofluorescence staining integrated with fluorescence insitu hybridization" (iFISH) was conducted along the diagnosis-metastasis-treatment-cellular MRD axis to longitudinally co-detect a full spectrum of intact CD31 - aneuploid tumor cells (TCs), CD31 + aneuploid tumor endothelial cells (TECs), viable and necrotic circulating TCs (CTCs) and circulating TECs (CTECs) expressing PD-L1, Ki67, p16 and Vimentin in unsliced specimens of the resected primary tumor, EM, dissected sentinel lymph nodes (SLNs) and peripheral blood in an early-stage melanoma patient. Numerous PD-L1 + aneuploid TCs and TECs were detected at the conventional safety EM (2 cm), quantitatively indicating the existence of a field cancerized EM for the first time. Contrary to highly heterogeneous PD-L1 expression and degrees of Chr8 aneuploidy in TCs and TECs in the primary lesions as well as CTCs and CTECs in peripheral blood, almost all TCs and TECs in SLNs and EM were homogeneously PD-L1 + haploid cells. Dynamic monitoring and cellular MRD assessment revealed that, in contrast to PD-L1 + CTCs being responsive to the immune checkpoint inhibitor (ICI-anti-PD-1), multiploid (≥pentasomy 8) PD-L1 + and Ki67 + CTECs were respectively resistant to ICI-sensitized T cells. In therapeutically stressed lymphatic and hematogenous metastatic cascades, stratified phenotypic and karyotypic profiling of iFISH tissue and liquid biopsied TCs, TECs, CTCs and CTECs in future large-cohort studies will enable appropriate re-specification of the optimal safety EM and distribution mapping of in-depth characterized, subcategorized target cells to help illustrate their metastatic relevance, ultimately improving risk stratification and clinical intervention of tumor progression, metastases, therapy resistance and cancer relapse., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cemiplimab has demonstrated relevant clinical activity in cutaneous squamous cell carcinoma (cSCC) but mechanisms of primary and acquired resistance to immunotherapy are still unknown. We collected clinical data from locally advanced and/or metastatic cSSC patients treated with cemiplimab in two Italian University centers. In addition, gene expression analysis by using Nanostring Technologies platform to evaluate 770 cancer- and immune-related genes on 20 tumor tissue samples (9 responders and 11 non-responders to cemiplimab) was performed. We enrolled 81 patients with a median age of 82 years. After 16.4 months of median follow-up, 12- and 24-months PFS were 53% and 42%, respectively; while 12- and 24-months OS were 71% and 61%, respectively. Treatment was well tolerated. Overall response rate (ORR) was 58%, with a disease control rate (DCR) of 77.8%. The difference between genes expressed in responder versus non-responder patient samples was substantial, particularly for genes involved in immune system regulation. Cemiplimab-resistant tumors were associated with over-expression of CCL-20 and CXCL-8. Cemiplimab confirmed efficacy and safety data in real-life cSCC patients. Overexpression of CCL-20 and CXCL-8 could represent biomarkers of lack of response to immunotherapy., Competing Interests: S.N. had travel grants from Amgen, Merck outside of the submitted works. D.C. had travel support from Sanofi, BMS, Merck serono outside of the submitted works. F.C. was advisory board for Amgen, Servier, MSD, Merck, Roche, Pfizer, Bayer, Pierre Fabre, Eisai outside of the submitted work. T.T. was advisory board for Amgen, MSD, Pierre Fabre, Roche, Merck outside of the submitted work. All remaining authors have no competing interests., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome is an autosomal-dominant disorder that results from a germline pathogenic variant in the fumarate hydratase (FH) gene on chromosome 1, characterised by renal cell carcinoma (RCC), cutaneous leiomyoma and uterine leiomyoma. Leiomyosarcomas are reported in less than 1% of those with HLRCC. We report a case of a man in his 30s who had a long-standing plaque excised from the left upper arm after undergoing a radical nephrectomy for a fumarate-deficient RCC, with histological exam revealing a grade 1 leiomyosarcoma. Genetic testing confirmed a heterozygous pathogenic variant in the FH gene. This is a rare case of leiomyosarcoma associated with HLRCC, and our patient remains under surveillance with interval abdominal imaging and skin examination. Leiomyosarcomas are difficult to distinguish clinically from their benign counterpart; therefore, histopathological examination is paramount with a low threshold for excision., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Neoadjuvant therapy improves recurrence-free survival (RFS) in resectable stage III cutaneous melanoma. However, accurately predicting individual recurrence risk remains a significant challenge. We investigated circulating tumour DNA (ctDNA) as a biomarker for recurrence in measurable stage IIIB/C melanoma patients undergoing neoadjuvant immunotherapy., Methods: Plasma samples were collected pre-neoadjuvant treatment, pre-surgery and/or six weeks post-surgery from 40 patients enrolled in the OpACIN-neo and PRADO clinical trials. Patients received two cycles of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) before surgery. Cell free DNA (cfDNA) underwent unbiased pre-amplification followed by tumour-informed mutation detection using droplet digital polymerase chain reaction (ddPCR) with the Bio-Rad QX600 PCR system., Results: Pre-treatment ctDNA was detectable in 19/40 (48%) patients. Among these, 17/19 (89%) zero-converted within six weeks of surgery and none recurred. Positive ctDNA post-surgery (N = 4), irrespective of pre-treatment ctDNA status, was 100% predictive of recurrence (sensitivity 44%, specificity 100%). Furthermore, ctDNA cleared prior to surgery in 7/9 (78%) patients who did not recur, warranting further investigation into ctDNA-guided surgical management., Conclusion: Post-surgery ctDNA positivity and zero-conversion are highly predictive of recurrence, offering a window for personalised modification of adjuvant therapy., (© 2024. The Author(s).)

Selenium and selenoproteins are closely related to melanoma progression. However, it is unclear how SELENOK affects lipid metabolism, endoplasmic reticulum stress (ERS), immune cell infiltration, survival, and prognosis in melanoma patients. Transcriptome data from melanoma patients was used to investigate SELENOK levels and their effect on prognosis, followed by an investigation of SELENOK's effects on immune cell infiltration. Furthermore, a risk model based on ERS, lipid metabolism, and immune-related genes was constructed, and its utility in melanoma prognosis was evaluated. Finally, the drug sensitivity of the risk model was analyzed to provide a reference for melanoma therapy. The results showed that melanoma with a high SELENOK level had a greater degree of immune cell infiltration and a better prognosis. Additionally, SELENOK was found to regulate ERS, lipid metabolism, and immune cell infiltration in melanoma. The risk model based on SELENOK signature genes successfully predicted the prognosis of melanoma, and the low-risk group exhibited a favorable immunological microenvironment. Furthermore, high-risk patients with melanoma were candidates for chemotherapy with RAS pathway inhibitors, whereas low-risk patients were more susceptible to routinely used chemotherapy medicines. In summary, SELENOK was shown to regulate ERS, lipid metabolism, and immune cell infiltration in melanoma, and SELENOK was positively associated with the prognosis of melanoma. The risk model based on SELENOK signature genes was valuable for melanoma prognosis and therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Melanoma is a malignant skin tumor with a high mortality rate. Regulatory T cells (Tregs) are immune cells with immunosuppressive roles, however, the precise mechanisms governing Treg involvement in melanoma remain enigmatic. Experimental findings unveiled different transcription factor switches between normal and tumor T cell, with heightened FOXP3 and BATF in the latter. These factors induced immunosuppressive molecules and Treg maintenance genes, polarizing tumor T cells into Tregs. Spatial transcriptomics illuminated the preferential settlement of Tregs at the melanoma periphery. Within this context, FOXP3 in Tregs facilitated direct enhancement of specific ligand gene expression, fostering communication with neighboring cells. Novel functional molecules bound to FOXP3 or BATF in Tregs, such as SPOCK2, SH2D2A, and ligand molecules ITGB2, LTA, CLEC2C, CLEC2D, were discovered, which had not been previously reported in melanoma Treg studies. Furthermore, we validated our findings in a large number of clinical samples and identified the Melanoma Treg-Specific Regulatory Tag Set (Mel TregS). ELISA analysis showed that the protein levels of Mel TregS in melanoma Tregs were higher than in normal Tregs. We then utilized SERS technology to measure the signal values of Mel TregS in exosome, and successfully discriminated between healthy individuals and melanoma patients, as well as early and late-stage patients. This approach significantly enhanced detection sensitivity. In sum, our research elucidated fresh insights into the mechanisms governing Treg self-maintenance and communication with surrounding cells in melanoma. We also introduced an innovative method for clinical disease monitoring through SERS technology., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare autosomal dominant syndrome caused by a germline mutation in the fumarate hydratase (FH) gene that manifests with cutaneous leiomyomas, uterine fibroids, and renal cell cancer (RCC). Patients with HLRCC-associated RCC (HLRCC-RCC) have aggressive clinical courses, but there is no standardized therapy for advanced HLRCC-RCC. In this study, we described a case of aggressive HLRCC in a 33-year-old female who exhibited a novel heterozygous germline insertion mutation in exon 8 of the FH gene (c.1126 C > T; p.Q376*). The patient underwent laparoscopic resection of the right kidney, but metastases appeared within 3 months after surgery. Histological staining of the resected tumor revealed high expression levels of programmed cell death-ligand 1 (PD-L1). Therefore, the patient was treated with immunotherapy. The patient achieved a partial response to immunotherapy, and the treatment of metastatic lesions has continued to improve. A thorough literature review pinpointed 76 historical cases of HLRCC-RCC that had undergone immunotherapy. From this pool, 46 patients were selected for this study to scrutinize the association between mutations in the FH gene and the effectiveness of immunotherapy. Our results indicate that immunotherapy could significantly improve the overall survival (OS) of patients with HLRCC-RCC. However, no influence of different mutations in the FH germline gene on the therapeutic efficacy of immunotherapy was observed. Therefore, our study suggested that immunotherapy was an effective therapeutic option for patients with HLRCC regardless of the type of FH germline mutation., (© 2024. The Author(s).)

The occurrence and progression of skin cutaneous melanoma (SKCM) is strongly associated with immune cells infiltrating the tumor microenvironment (TME). This study examined the expression, prognosis, and immune relevance of SIGLEC9 in SKCM using multiple online databases. Analysis of the GEPIA2 and Ualcan databases revealed that SIGLEC9 is highly expressed in SKCM, and patients with high SIGLEC9 expression had improved overall survival (OS). Furthermore, the mutation rate of SIGLEC9 in SKCM patients was found to be 5.41%, the highest observed. The expression of SIGLEC9 was positively correlated with macrophages, neutrophils and B cells, CD8 + T cells, CD4 + T cells, and dendritic cells, according to TIMER. Based on TCGA-SKCM data, we verified that high SIGLEC9 expression is closely associated with a good prognosis for SKCM patients, including overall survival, progression-free interval, and disease-specific survival. This positive prognosis could be due to the infiltration of immune cells into the TME. Additionally, our analysis of single-cell transcriptome data revealed that SIGLEC9 not only played a role in the normal skin immune microenvironment, but is also highly expressed in immune cell subpopulations of SKCM patients, regulating the immune response to tumors. Our findings suggest that the close association between SIGLEC9 and SKCM prognosis is primarily mediated by its effect on the tumor immune microenvironment., (© 2024. The Author(s).)

Given the importance of peroxisome proliferator-activated receptor (PPAR)-gamma in epidermal inflammation and carcinogenesis, we analyzed the transcriptomic changes observed in epidermal PPARγ-deficient mice ( Pparg -/- epi ). A gene set enrichment analysis revealed a close association with epithelial malignancy, inflammatory cell chemotaxis, and cell survival. Single-cell sequencing of Pparg -/- epi mice verified changes to the stromal compartment, including increased inflammatory cell infiltrates, particularly neutrophils, and an increase in fibroblasts expressing myofibroblast marker genes. A comparison of transcriptomic data from Pparg -/- epi and publicly available human and/or mouse actinic keratoses (AKs) and cutaneous squamous cell carcinomas (SCCs) revealed a strong correlation between the datasets. Importantly, PPAR signaling was the top common inhibited canonical pathway in AKs and SCCs. Both AKs and SCCs also had significantly reduced PPARG expression and PPARγ activity z-scores. Smaller reductions in PPARA expression and PPARα activity and increased PPARD expression but reduced PPARδ activation were also observed. Reduced PPAR activity was also associated with reduced PPARα/RXRα activity, while LPS/IL1-mediated inhibition of RXR activity was significantly activated in the tumor datasets. Notably, these changes were not observed in normal sun-exposed skin relative to non-exposed skin. Finally, Ppara and Pparg were heavily expressed in sebocytes, while Ppard was highly expressed in myofibroblasts, suggesting that PPARδ has a role in myofibroblast differentiation. In conclusion, these data provide strong evidence that PPARγ and possibly PPARα represent key tumor suppressors by acting as master inhibitors of the inflammatory changes found in AKs and SCCs.

Competing Interests: Conflicts of interest The authors declare no conflicts of interest.

The transcription factor ΔNp63 plays a pivotal role in maintaining the integrity of stratified epithelial tissues by regulating the expression of distinct target genes involved in lineage specification, cell stemness, cell proliferation and differentiation. Here, we identified the ABC transporter subfamily member ABCC1 as a novel ΔNp63 target gene. We found that in immortalized human keratinocytes and in squamous cell carcinoma (SCC) cells, ∆Np63 induces the expression of ABCC1 by physically occupying a p63-binding site (p63 BS) located in the first intron of the ABCC1 gene locus. In cutaneous SCC and during the activation of the keratinocyte differentiation program, ∆Np63 and ABCC1 levels are positively correlated raising the possibility that ABCC1 might be involved in the regulation of the proliferative/differentiative capabilities of squamous tissue. However, we did not find any gross alteration in the structure and morphology of the epidermis in humanized hABCC1 knock-out mice. Conversely, we found that the genetic ablation of ABCC1 led to a marked reduction in inflammation-mediated proliferation of keratinocytes, suggesting that ABCC1 might be involved in the regulation of keratinocyte proliferation upon inflammatory/proliferative signals. In line with these observations, we found a significant increase in ABCC1 expression in squamous cell carcinomas (SCCs), a tumor type characterized by keratinocyte hyper-proliferation and a pro-inflammatory tumor microenvironment. Collectively, these data uncover ABCC1 as an additional ∆Np63 target gene potentially involved in those skin diseases characterized by dysregulation of proliferation/differentiation balance.

Objective: To investigate the clinicopathological, immunohistochemical and molecular characteristics of cutaneous ALK-rearranged Spitz melanocytoma. Methods: Two cases of cutaneous ALK-rearranged Spitz melanocytoma from outside hospital consultations in Department of Pathology, Affiliated Cancer Hospital of Fudan University in August 2020 and in Shanghai Ackermann Medical Laboratory in June 2022 were collected. The clinicopathological features, immunophenotypes and molecular profiles of two patients with cutaneous Spitzoid melanocytic tumor harboring ALK-rearrangement were analyzed. The literatures were reviewed. Results: The study included an 8-year-old boy and an 11-year-old girl, who presented with a polypoid lesion in the skin of right thigh and left auricle measuring 1.0 cm and 1.2 cm, respectively. Histologically, they were composed of medium to large-sized epithelioid to plump spindle cells, arranged in nested, plexiform or fascicular patterns in the superficial dermis. The neoplastic cells had abundant eosinophilic cytoplasm with round to ovoid vesicular nuclei containing prominent eosinophilic nucleoli. One case showed mild to moderate nuclear pleomorphism and mitotic activity (average, 2/mm 2 ). Immunohistochemically, the epithelioid and plump spindle cells showed diffuse and strong staining of S-100 protein, SOX10, and ALK (D5F3 and 1A4), but did not express HMB45, PNL2 and MiTF. ALK-rearrangement was detected by fuorescence in situ hybridization in both cases. Subsequent next generation sequence (NGS) analysis identified KANK1::ALK and TPM3:ALK fusions. At 34 and 14 months after surgical resection, both patients remained well with no signs of recurrence or metastasis. Conclusions: ALK-rearranged Spitz melanocytoma represents a morphologically and genetically distinct subset of Spitz melanocytoma, characterized clinically by predilection in children and adolescents, with Spitzoid morphology in plexiform pattern, positive immunohistochemical stains, and rearrangement of ALK. As some cases show atypical features and high mitotic activity, a distinction from Spitz melanoma is warranted.

Objective: To investigate the clinical, cytomorphology, immunocytochemical and molecular features of metastatic melanoma in serosal cavity effusion. Methods: Cytological specimens of 14 patients with melanoma in the chest and abdomen were collected from 2017 to 2023, at the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. SOX10, S-100 protein, PRAME, BRAF V600E, HMB45, and Melan A were detected by immunocytochemical methods. Fourteen cases were tested for routine antibody combinations, including Claudin4, HEG1, Calretinin, CD68, etc. Four of the patients had biopsy or surgical samples of metastatic solid lesions of primary sites, and further next-generation sequencing (NGS) or amplification refractory mutation system (ARMS)-PCR molecular test was performed. In addition, 30 cases of serosal effusion samples were collected as control groups (10 cases of benign mesothelial cell reactive hyperplasia, 10 cases of mesothelioma, and 10 cases of metastatic lung adenocarcinoma). Results: Among the 14 cases of melanoma, there were 7 males and 7 females, with ages ranging from 35 to 86 years, and an average age of 57 years, there 10 cases aged ≥50 years. The tumor cells in the serosal effusion varied in morphology and degree of atypia. SOX10 was positive in all 14 cases (14/14), S-100 protein was positive in 10 cases (10/14), PRAME was positive in 12 cases (12/14), BRAF V600E was positive in 10 cases (10/14), HMB45 was positive in 12 cases (12/14), and Melan A was positive in 13 cases (13/14). In 4 patients with histological correlation, the cytological and histological expression of SOX10, BRAF V600E, and PRAME was positive in all 4 cases (4/4); S-100 protein was positive in 2 cases (2/4); and HMB45 and Melan A were positive in 3 cases (3/4). Using NGS or ARMS-PCR, missense mutations of BRAF V600E were detected in all 4 patients; TERT promoter mutations was detected in 1 case; and CDKN2A terminating mutations and MSI1 deletion mutations were detected in the other case. SOX10, S-100, HMB45, Melan A, PRAME and BRAF V600E were all negative in 30 control samples of serosal cavity effusion. Conclusion: By observing the morphology of tumor cells, immunocytochemical test of several combination markers, especially the expression of SOX10, BRAF V600E and PRAME, can help to improve the positive diagnosis rate of melanoma in serous cavity effusion.

Differentiation of murine epidermal stem/progenitor cells involves the permanent withdrawal from the cell cycle, the synthesis of various protein and lipid components for the cornified envelope, and the controlled dissolution of cellular organelles and nuclei. Deregulated epidermal differentiation contributes to the development of various skin diseases, including skin cancers. With a genome-wide shRNA screen, we identified vesicle-associated membrane protein 2 (VAMP2) as a critical factor involved in skin differentiation. Deletion of VAMP2 leads to aberrant skin stratification and enucleation in vivo. With quantitative proteomics, we further identified an autophagy protein, focal adhesion kinase family interacting protein of 200 kDa (FIP200), as a binding partner of VAMP2. Additionally, we showed that both VAMP2 and FIP200 are critical for murine keratinocyte enucleation and epidermal differentiation. Loss of VAMP2 or FIP200 enhances cutaneous carcinogenesis in vivo. Together, our findings identify important molecular mechanisms underlying epidermal differentiation and skin tumorigenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The keratinocyte carcinomas, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC), are the most common cancers in humans. Recently, an increasing body of literature has investigated the role of miRNAs in keratinocyte carcinoma pathogenesis, progression and their use as therapeutic agents and targets, or biomarkers. However, there is very little consistency in the literature regarding the identity of and/or role of individual miRNAs in cSCC (and to a lesser extent BCC) biology. miRNA analyses that combine clinical evidence with experimental elucidation of targets and functional impact provide far more compelling evidence than studies purely based on clinical findings or bioinformatic analyses. In this study, we review the clinical evidence associated with miRNA dysregulation in KCs, assessing the quality of validation evidence provided, identify gaps, and provide recommendations for future studies based on relevant studies that investigated miRNA levels in human cSCC and BCC. Furthermore, we demonstrate how miRNAs contribute to the regulation of a diverse network of cellular functions, and that large-scale changes in tumor cell biology can be attributed to miRNA dysregulation. We highlight the need for further studies investigating the role of miRNAs as communicators between different cell types in the tumor microenvironment. Finally, we explore the clinical benefits of miRNAs as biomarkers of keratinocyte carcinoma prognosis and treatment.

The Basal Cell Carcinoma (BCC) is a sort of unique tumour due to its combined peculiar histological features and clinical behaviour, such as the constant binary involvement of the epithelium and the stroma, the virtual absence of metastases and the predilection of specific anatomical sites for both onset and spread. A potential correlation between the onset of BCC and a dysembryogenetic process has long been hypothesised. A selective investigation of PubMed-indexed publications supporting this theory retrieved 64 selected articles published between 1901 and 2024. From our analysis of the literature review, five main research domains on the dysembryogenetic pathogenesis of BCC were identified: (1) The correlation between the topographic distribution of BCC and the macroscopic embryology, (2) the correlation between BCC and the microscopic embryology, (3) the genetic BCC, (4) the correlation between BCC and the hair follicle and (5) the correlation between BCC and the molecular embryology with a specific focus on the Hedgehog signalling pathway. A large amount of data from microscopic and molecular research consistently supports the hypothesis of a dysembryogenetic pathogenesis of BCC. Such evidence is promoting advances in the clinical management of this disease, with innovative targeted molecular therapies on an immune modulating basis being developed.

Background: Cutaneous squamous cell carcinoma (cSCC) is a fatal disease characterized by metabolic dysregulation. The role of ephrin type-B receptor 2 (ephrin-B2), a crucial molecule in cancer cell biology, in regulating glycolysis and cell proliferation of cSCC is not well understood. This study aimed to investigate the biological pathways by which ephrin-B2 impacts the glycolysis and cell proliferation of cSCC., Methods: Ephrin-B2 expression levels in cSCC were determined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Ephrin-B2 expression in cSCC cells was manipulated using overexpression and knockdown approaches. A series of in vitro assays, such as cell counting kit-8 (CCK-8), Transwell assay, immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), qRT-PCR, and Western blotting, were employed to delineate the biological roles of ephrin-B2/pyruvate kinase muscle isoenzyme 2 (PKM2)/hypoxia-inducible factor 1 alpha (HIF-1α) in proliferation, migration, invasion, and glucose metabolism of cSCC., Results: This study highlights an upregulation of ephrin-B2 expression in cSCC. Knockdown of ephrin-B2 significantly suppressed the proliferation, migration, invasion, and glucose metabolism of cSCC cells. Moreover, ephrin-B2 expression was upregulated under hypoxic conditions. At the molecular level, ephrin-B2 knockdown resulted in the downregulation of PKM2 and HIF-1α expression. Additionally, the overexpression of PKM2 or HIF-1α successfully rescued the diminished proliferation, migration, invasion and glucose metabolism induced by ephrin-B2 knockdown in cSCC cells., Conclusion: These findings suggest that ephrin-B2 suppression may hinder cSCC cell proliferation and glycolytic metabolism, potentially via the PKM2/HIF-1α axis modulation.