Decoding the Molecular Mechanisms of BRAF V600E-Induced Nevi Formation.

Melanocytes derived from neural crest cells harbor the BRAF V600E mutation, which is the predominant driver of nevus formation in humans. This mutation leads to malignant cell proliferation and subsequent cell cycle arrest, culminating in oncogene-induced senescence and nevus development. Nevertheless, emerging evidence has highlighted the heterogeneity of cellular senescence markers in BRAF V600E-induced senescent melanocytes. Moreover, the capacity of melanocytes within nevi to regain their proliferative ability raises questions about the molecular mechanisms by which BRAF V600E, via the mitogen-activated protein kinase signaling pathway, triggers nevus formation. This study provides an overview and discussion of the molecular mechanisms underpinning BRAF V600E-induced melanocyte nevus formation and the relevant animal models employed for their elucidation. It also highlights the significance of elucidating dynamic changes in cytoplasmic and nuclear substrates that interact with phosphorylated extracellular signal-regulated protein kinases 1 and 2 and underscores the value of using targeted BRAF V600E animal models created through gene editing technologies.

PRAME and Historical Immunohistochemical Antibodies Ki-67, P16, and HMB-45 in Ambiguous Melanocytic Tumors.

ABSTRACT: Ambiguous melanocytic lesions/tumors (AMLs) can be simply described as melanocytic neoplasms that cannot be differentiated as either a melanoma or a nevus. Preferentially expressed antigen in melanoma (PRAME) is a novel antibody that can help differentiate between nevi and melanomas. However, its usefulness remains controversial in AMLs. The aim of this study was to demonstrate the importance of PRAME and diagnostic auxiliary antibodies (Ki-67, p16, HMB-45) in the diagnosis of melanocytic lesions, especially in AMLs. This study included 52 ambiguous melanocytic lesions, 40 nevi, and 40 melanomas. All immunohistochemical studies were performed automatically using the Universal Alkaline Phosphatase Red Detection Kit. Different analytic approaches were used for each antibody based on the literature. Statistically, the multinomial forward stepwise elimination logistic regression analysis was used to create a statistical model to predict the diagnosis of melanocytic lesions based on clinical, morphological, and immunohistochemical data. PRAME positivity was very strong and diffuse in the melanoma group and statistically significantly higher than that of the AML and nevus groups. There was no statistically significant difference between the nevus and AML groups. The Ki-67 proliferation index and HMB-45 staining pattern provided valuable indications for distinguishing between these 3 groups. The P16 antibody was limited in supporting the differential diagnosis. Our statistical model showed that a high mitosis count, central pagetoid spread, and PRAME positivity increased the probability of melanoma against an AML diagnosis. This study showed the advantages of evaluating the PRAME antibody together with morphological features and other immunohistochemical markers (Ki-67 and HMB-45) in the differential diagnosis of melanocytic lesions.

Comparative Quantitative Proteomic Analysis of Melanoma Subtypes, Nevus-Associated Melanoma, and Corresponding Nevi.

A substantial part of cutaneous malignant melanomas develops from benign nevi. However, the precise molecular events driving the transformation from benign to malignant melanoma are not well-understood. We used laser microdissection and mass spectrometry to analyze the proteomes of melanoma subtypes, including superficial spreading melanomas (n = 17), nodular melanomas (n = 17), and acral melanomas (n = 15). Furthermore, we compared the proteomes of nevi cells with those of melanoma cells within the same specimens (nevus-associated melanoma (n = 14)). In total, we quantified 7935 proteins. Despite the genomic and clinical differences of the melanoma subtypes, our analysis revealed relatively similar proteomes, except for the upregulation of proteins involved in immune activation in nodular melanomas versus acral melanomas. Examining nevus-associated melanoma versus nevi, we found 1725 differentially expressed proteins (false discovery rate < 0.05). Among these proteins were 140 that overlapped with cancer hallmarks, tumor suppressors, and regulators of metabolism and cell cycle. Pathway analysis indicated aberrant activation of the phosphoinositide 3-kinase-protein kinase B-mTOR pathways and the Hippo-YAP pathway. Using a classifier, we identified six proteins capable of distinguishing melanoma from nevi samples. Our study represents a comprehensive comparative analysis of the proteome in melanoma subtypes and associated nevi, offering insights into the biological behavior of these distinct entities.

Unusual conjunctival melanocytic proliferations. Report of five cases and review of the literature.

Indeterminate melanocytic proliferations of the conjunctiva have both benign and malignant features that previously made these lesions nearly impossible to categorize in existing classification schemes. With the evolution of immunohistochemistry and molecular genetics, however, subclassifications have emerged that allow for a more tailored diagnosis and management. These conjunctival melanocytic proliferations include deep penetrating nevus, granular cell nevus, and nevoid melanoma. There remains a small subset of conjunctival melanocytic proliferations that defy precise characterization as nevi, primary acquired melanosis, or melanomas despite currently available ancillary diagnostic modalities and remain indeterminate. We highlight these unusual types of nevi and melanomas, with an update on their morphologic, immunohistochemical, and molecular genetic characteristics.

Immunohistochemistry for PRAME in Dermatopathology.

ABSTRACT: Preferentially expressed antigen in melanoma (PRAME) is a tumor-associated antigen first identified in a melanoma patient and found to be expressed in most melanomas as well as in variable levels in other malignant neoplasms of epithelial, mesenchymal, or hematolymphoid lineage. Detection of PRAME expression in formalin-fixed paraffin-embedded tissue is possible by immunohistochemistry (IHC) with commercially available monoclonal antibodies. In situ and invasive melanoma frequently show a diffuse pattern of nuclear PRAME immunoreactivity which contrasts with the infrequent and typically nondiffuse staining seen in nevi. In many challenging melanocytic tumors, results of PRAME IHC and other ancillary tests correlate well, but not always: The tests are not interchangeable. Most metastatic melanomas are positive for PRAME, whereas nodal nevi are not. Numerous studies on PRAME IHC have become available in the past few years with results supporting the value of PRAME IHC as an ancillary tool in the evaluation of melanocytic lesions and providing insights into limitations in sensitivity and specificity as well as possible pitfalls that need to be kept in mind by practicing pathologists.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression.

Human melanocytic nevi (moles) result from a brief period of clonal expansion of melanocytes. As a cellular defensive mechanism against oncogene-induced hyperplasia, nevus-resident melanocytes enter a senescent state of stable cell cycle arrest. Senescent melanocytes can persist for months in mice and years in humans with a risk to escape the senescent state and progress to melanoma. The mechanisms providing prolonged survival of senescent melanocytes remain poorly understood. Here, we show that senescent melanocytes in culture and in nevi express high level of the anti-apoptotic BCL-2 family member BCL-W but remain insensitive to the pan-BCL-2 inhibitor ABT-263. We demonstrate that resistance to ABT-263 is driven by mTOR-mediated enhanced translation of another anti-apoptotic member, MCL-1. Strikingly, the combination of ABT-263 and MCL-1 inhibitors results in synthetic lethality to senescent melanocytes, and its topical application sufficient to eliminate nevi in male mice. These data highlight the important role of redundant anti-apoptotic mechanisms for the survival advantage of senescent melanocytes, and the proof-of-concept for a non-invasive combination therapy for nevi removal.

[Application of immunohistochemical staining of PRAME in differential diagnosis between melanoma and melanocytic nevus].

Objective: To investigate the diagnostic value of preferentially expressed antigen in melanoma (PRAME) in differential diagnosis of benign and malignant cutaneous melanocytic lesions. Methods: Fifty-nine cases of melanoma (50 cases of skin primary melanoma, and 9 cases of metastatic melanoma) and 48 cases of melanocytic nevus (40 cases of common nevus and 8 cases of dysplastic nevus) were subject to PRAME immunohistochemistry staining.The difference of PRAME expression between melanoma and melanocytic nevus was analyzed. Results: Among the 50 patients with primary cutaneous melanoma, there were 23 males and 27 females ranging in age from 33 to 87 years (average age 62.4 years, median age 64.5 years). Among the 9 metastatic melanoma there were 7 males and 2 females ranging in age from 40 to 82 years (average age 64 years, median age 65 years). Twenty-six cases (26/50, 52.0%) of cutaneous primary melanoma and 4 cases (4/9) of metastatic melanoma showed diffuse positive PRAME staining. 40 cases (40/40, 100%) of common nevus and 8 (8/8) cases of dysplastic nevus were PRAME negative. Compared with melanocytic nevus group, the melanoma group included more cases with diffuse positive PRAME staining (P<0.05). The sensitivity and specificity of using PRAME to differentiate primary cutaneous melanoma from melanocytic nevus in the cohort is 52.0% and 100%. Conclusions: There is a significant difference in the expression of PRAME between melanoma and melanocytic nevus.Thus, PRAME can be used as an auxiliary diagnostic tool for differentiating benign from malignant cutaneous lesions.

Inactivation of the Hippo tumor suppressor pathway promotes melanoma.

Melanoma is commonly driven by activating mutations in the MAP kinase BRAF; however, oncogenic BRAF alone is insufficient to promote melanomagenesis. Instead, its expression induces a transient proliferative burst that ultimately ceases with the development of benign nevi comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that restrain nevus melanocyte proliferation remain poorly understood. Here we utilize cell and murine models to demonstrate that oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway, both in melanocytes in vitro and nevus melanocytes in vivo. Mechanistically, we show that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome doubling events, which independently reduce RhoA activity to promote Hippo activation. We also demonstrate that functional impairment of the Hippo pathway enables oncogenic BRAF-expressing melanocytes to bypass nevus formation and rapidly form melanomas. Our data reveal that the Hippo pathway enforces the stable arrest of nevus melanocytes and represents a critical barrier to melanoma development.

Preferentially expressed antigen in melanoma and p16 expression in acral melanocytic neoplasms.

Acral melanocytic neoplasms often pose diagnostic difficulty. Preferentially expressed antigen in melanoma (PRAME) expression and loss of p16 expression have diagnostic utility in melanocytic tumors. We examined PRAME and p16 expression in 30 acral melanocytic neoplasms (n = 11 nevi; n = 2 dysplastic nevi; n = 7 Spitz nevi; n = 10 acral melanomas). PRAME was scored as % positive nuclei: negative = 0%; 1% to 25% = 1+; 25% to 50% = 2+; 50% to 75% = 3+, or positive: 75% to 100% = 4+. p16 expression was defined as retained (homogeneous or checkerboard) or lost (complete or partial/regionally). PRAME expression was negative in all benign, dysplastic, and Spitz nevi. Conversely, all acral melanomas were diffusely (4+) positive for PRAME expression. p16 expression was retained in all benign acral nevi (8/11 homogeneous, 3/11 checkerboard), completely lost in one dysplastic nevus, and retained in all acral Spitz nevi (3/7 homogeneous, 4/7 checkerboard). p16 was retained in five of 10 acral melanomas (3/10 homogeneous; 2/10 checkerboard), and negative in five of 10 acral melanomas (absent in 3/10, partially lost in 2/10). Our data suggest that 4+ PRAME expression is highly sensitive and specific in the setting of acral melanomas and is a more predictive diagnostic tool compared with p16 immunohistochemistry.

SSRP1 Worsens Malignant Melanoma Progression by Activating MAPKs Pathway.

OBJECTIVE: Structure-specific recognition protein 1 (SSRP1) is highly expressed in a variety of tumors and promotes cell proliferation and migration. Malignant melanoma is a highly malignant skin malignancy with low morbidity and high mortality. The role of SSRP1 in malignant melanoma is still unclear. Thus, this study is intended to investigate the role of SSRP1 in malignant melanoma and reveal the related mechanisms. METHODS: Western blots and immunohistochemistry assays were used to determine the expression of SSRP1 in benign nevi tissues and malignant melanoma tissues. The si-SSRP1 was used to knockdown the expression level of SSRP1 in A375 cells. Cell proliferation was assessed by MTT assay. Wound healing and Transwell assay were performed for detected cell migratory and invasive activities, respectively. Besides, the expression levels of epithelial-mesenchymal transition (EMT) markers and MAPKs signaling pathway were measured by western blot. RESULTS: The results showed that SSRP1 was highly expressed in malignant melanoma tissues and cells, and its expression in metastatic melanoma tissues was significantly higher than that in primary melanoma. Besides, high expression level of SSRP1 was accompanied with poor prognosis in malignant melanoma patients. SSRP1 knockdown inhibited the melanoma cell proliferation, migration, and invasion. Besides, SSRP1 knockdown inhibited the process of EMT by upregulating E-cadherin, and downregulating N-cadherin and vimentin. Further studies revealed that SSRP1 silencing affected MAPK signaling pathway and reduced its phosphorylation activity in melanoma cells. CONCLUSIONS: These results suggested that SSRP1 may promote the proliferation, migration, and invasion of melanoma cells through MAPK signaling pathway. SSRP1 is closely related to the malignancy of melanoma and may be a potential target for its clinical treatment.

Prevalence of nevi, atypical nevi, and lentigines in relation to tobacco smoking.

BACKGROUND: Melanocytic nevi have a complex evolution influenced by several endogenous and exogenous factors and are known risk factors for malignant melanoma. Interestingly, tobacco use seems to be inversely associated with melanoma risk. However, the association between tobacco use and nevi and lentigines has not yet been evaluated. METHODS: We investigated the prevalence of nevi, atypical nevi, and lentigines in relation to tobacco smoking in a cohort of 59 smokers and 60 age- and sex-matched nonsmokers, using a questionnaire and performing a total body skin examination by experts. RESULTS: No significant differences were detected between smokers and nonsmokers in the numbers of nevi, atypical nevi, and lentigines in sun-exposed areas (p = 0.966, 0.326, and 0.241, respectively) and in non-sun-exposed areas (p = 0.095, 0.351, and 0.546, respectively). CONCLUSION: Our results revealed no significant differences in the prevalence of nevi, atypical nevi, and lentigines between smokers and nonsmokers in sun-exposed and non-sun-exposed areas.

Post Zygotic, Somatic, Deletion in KERATIN 1 V1 Domain Generates Structural Alteration of the K1/K10 Dimer, Producing a Monolateral Palmar Epidermolytic Nevus.

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.

Coexistence of three nevus lipomatosus cutaneus superficialis with typical, cutaneous adenolipoma-like, and dermal spindle cell lipoma-like histopathological features in a patient.

We report an unique case of a patient who showed coexistence of three nevus lipomatosus cutaneus superficialis (NLCS) with typical, cutaneous adenolipoma (AL)-like, and dermal spindle cell lipoma (SCL)-like histopathological features. A 53-year-old woman presented with a 20-year history of skin-colored and slightly elevated nodules. These lesions were separately located on the lateral side (lesion 1) and medial side (lesion 2) of her left buttock and on her right thigh (lesion 3). Microscopically, all were ill-defined dermal lesions with some subcutaneous involvement and were mostly composed of mature adipocytes. The adipocytes formed small aggregates around blood vessels in the upper dermis. Lesions 1, 2, and 3 were diagnosed as NLCS, and additional features were recognized in lesions 2 and 3. Lesion 2 revealed eccrine glands and ducts amongst the lipomatous component, as seen in cutaneous AL. Lesion 3 had scattered CD34-positive spindle cells, which is representative of dermal SCL. These appearances were considered to be on the morphological spectrum of NLCS. In all three lesions, CD34-positive cells proliferated between the upper dermal blood vessels and their peripheral mature adipocytes. This pathological finding could be principal in NLCS and might be associated with its pathogenesis.

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma.

BACKGROUND: Activating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown. METHODS: We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. RESULTS: 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. CONCLUSIONS: Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma.

The utility of PRAME staining in identifying malignant transformation of melanocytic nevi.

BACKGROUND: PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemical (IHC) staining is used to aid melanoma diagnosis. PRAME expression in nevus-associated melanoma (NAM) has not been evaluated. METHODS: PRAME IHC was applied to cases of NAM; staining for each population of melanocytes (benign and malignant) was graded based on the percentage of labeled cells. No labeling was graded 0, 1% to 25% labeling was 1+, 26% to 50% was 2+, 51% to 75% was 3+, and >76% was 4+. RESULTS: Thirty-six cases were reviewed. Sixty-seven percent (24/36) of melanomas were PRAME positive (4+) while no (0/36) nevi showed 4+ positivity. Eighty-one percent (29/36) of nevi were completely PRAME negative compared to 17% (6/36) of melanomas. In 67% of cases (24/36) PRAME differentiated between benign and malignant melanocyte populations. CONCLUSIONS: We identified a high rate (67%) of differential PRAME staining in adjacent benign and malignant melanocyte populations in NAM. In PRAME positive (4+) melanomas, PRAME differentiates 100% (24/24) of benign and malignant melanocyte populations. When 4+ staining is used as the threshold for positivity, PRAME staining has a sensitivity of 67% (24/36) and a specificity of 100% (36/36). These results support PRAME IHC can assist in distinguishing melanocyte populations in melanoma arising within nevi.

Expression profiles of proton-sensing G-protein coupled receptors in common skin tumors.

The proton-sensing GPCRs (pH-GPCRs) GPR4 (GPR19), TDAG8 (GPR65, T-cell death associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1), and G2A (GPR132, G2 accumulation protein) are involved in sensing and transducing changes in extracellular pH (pHe). Extracellular acidification is a central hallmark of solid cancer. pH-GPCR function has been associated with cancer cell proliferation, adhesion, migration and metastasis, as well as with modulation of the immune system. Little is known about the expression levels and role of pH-GPCRs in skin cancer. To better understand the functions of pH-GPCRs in skin cancer in vivo, we examined the expression-profiles of GPR4, TDAG8, OGR1 and G2A in four common skin tumors, i.e. squamous cell carcinoma (SCC), malignant melanoma (MM), compound nevus cell nevi (NCN), basal cell carcinoma (BCC). We performed immunohistochemistry and immunofluorescence staining on paraffin-embedded tissue samples acquired from patients suffering from SCC, MM, NCN or BCC. We show the expression of pH-GPCRs in four common skin cancers. Different expression patterns in the investigated skin cancer types indicate that the different pH-GPCRs may have distinct functions in tumor progression and serve as novel therapeutic targets.

Neutralization of HSF1 in cells from PIK3CA-related overgrowth spectrum patients blocks abnormal proliferation.

PIK3CA-related overgrowth spectrum is caused by mosaicism mutations in the PIK3CA gene. These mutations, which are also observed in various types of cancer, lead to a constitutive activation of the PI3K/AKT/mTOR pathway, increasing cell proliferation. Heat shock transcription factor 1 (HSF1) is the major stress-responsive transcription factor. Recent findings indicate that AKT phosphorylates and activates HSF1 independently of heat-shock in breast cancer cells. Here, we aimed to investigate the role of HSF1 in PIK3CA-related overgrowth spectrum. We observed a higher rate of proliferation and increased phosphorylation of AKT and p70S6K in mutant fibroblasts than in control cells. We also found elevated phosphorylation and activation of HSF1, which is directly correlated to AKT activation. Specific AKT inhibitors inhibit HSF1 phosphorylation as well as HSF1-dependent gene transcription. Finally, we demonstrated that targeting HSF1 with specific inhibitors reduced the proliferation of mutant cells. As there is currently no curative treatment for PIK3CA-related overgrowth spectrum, our results identify HSF1 as a new potential therapeutic target.