Melanoma Markers Research Articles

630 Cdk2: A marker for invasive melanoma

Background: Although cyclin-dependent kinase 2 (CDK2) is known to be upregulated in certain cancers, its spatial expression pattern in melanocytic tumors has remained unknown. Objective: To characterize CDK2 expression in benign and malignant melanocytic proliferations. Methods: We performed CDK2 immunohistochemistry (IHC) on normal skin and a tumor panel comprising over 200 congenital nevi, dysplastic nevi, melanomas in situ, and invasive melanomas. We recorded their staining patterns, intensities, and compared these to expression of melanoma marker PRAME (Preferentially expressed Antigen in Melanoma).

Bioinformatic analysis identifies epidermal development genes that contribute to melanoma progression.

Several diagnostic and prognostic markers for melanoma have been identified in last few years. However, their actual contribution to melanoma progression have not been investigated in detail. This study was aimed to identify genes, biological processes, and signaling pathways implicated in melanoma progression by applying bioinformatics analysis. We identified nine differentially expressed genes (DEGs) (IL36RN, KRT6A, KRT6B, KRT16, S100A7, SPRR1A, SPRR1B, SPRR2B, and KLK7) that were upregulated in primary melanoma compared with metastatic melanoma in all five datasets analyzed. All these genes except IL36RN, both form a protein-protein interaction network and have cellular functions associated with constitutive processes of keratinocytes. Thus, they were generically termed Epidermal Development and Cornification (EDC) genes. The differential expression of these genes in primary and metastatic melanoma was confirmed in the TCGA-SKCM cohort. High expression of the EDC genes correlated with reduced tumor thickness in primary melanoma and shorter survival in metastatic melanoma. Analysis of DEGs from primary melanoma patients displaying high or low expression of all eight EDC revealed that the upregulated genes are enriched in biological process related to cell migration, extracellular matrix organization, invasion, and Epithelial-Mesenchymal Transition. Further analysis of enriched curated oncogenic genesets together with RPPA data of phosphorylated proteins revealed the activation of MEK, ATF2, and EGFR pathways in tumors displaying high expression of EDC genes. Thus, EDC genes may contribute to melanoma progression by promoting the activation of MEK, ATF2, and EGFR pathways together with biological processes associated with tumor aggressiveness.

Knockdown of Lamin B1 and the Corresponding Lamin B Receptor Leads to Changes in Heterochromatin State and Senescence Induction in Malignant Melanoma.

Modifications in nuclear structures of cells are implicated in several diseases including cancer. They result in changes in nuclear activity, structural dynamics and cell signalling. However, the role of the nuclear lamina and related proteins in malignant melanoma is still unknown. Its molecular characterisation might lead to a deeper understanding and the development of new therapy approaches. In this study, we analysed the functional effects of dysregulated nuclear lamin B1 (LMNB1) and its nuclear receptor (LBR). According to their cellular localisation and function, we revealed that these genes are crucially involved in nuclear processes like chromatin organisation. RNA sequencing and differential gene expression analysis after knockdown of LMNB1 and LBR revealed their implication in important cellular processes driving ER stress leading to senescence and changes in chromatin state, which were also experimentally validated. We determined that melanoma cells need both molecules independently to prevent senescence. Hence, downregulation of both molecules in a BRAFV600E melanocytic senescence model as well as in etoposide-treated melanoma cells indicates both as potential senescence markers in melanoma. Our findings suggest that LMNB1 and LBR influence senescence and affect nuclear processes like chromatin condensation and thus are functionally relevant for melanoma progression.

Comprehensive Analyses of miRNAs Revealed miR-92b-3p, miR-182-5p and miR-183-5p as Potential Novel Biomarkers in Melanoma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are important mediators in the intercellular communication, influencing the function and phenotype of different cell types within the tumor micro-milieu and thus promote tumor progression. Since EVs safely transport packages of proteins, lipids and also nucleic acids such as miRNAs, EVs and their cargo can serve as diagnostic and prognostic markers. Therefore, the aim of this study was to investigate EV embedded miRNAs specific for melanoma, which could serve as potential biomarkers. In contrast to previous studies, we not only analysed miRNAs from EVs, but also included the miRNA profiles from the EV-secreting cells to identify candidates as suitable biomarkers. While the characterization of EVs derived from normal melanocytes and melanoma cells showed largely comparable properties with regard to size distribution and expression of protein markers, the NGS analyses yielded marked differences for several miRNAs. While miRNA load of EVs derived from normal human epidermal melanocytes (NHEMs) and melanoma cells were very similar, they were highly different from their secreting cells. By comprehensive analyses, six miRNAs were identified to be enriched in both melanoma cells and melanoma cell-derived EVs. Of those, the accumulation of miR-92b-3p, miR-182-5p and miR-183-5p in EVs could be validated in vitro. By functional network generation and pathway enrichment analysis we revealed an association with different tumor entities and signaling pathways contributing melanoma progression. Furthermore, we found that miR-92b-3p, miR-182-5p and miR-183-5p were also enriched in EVs derived from serum of melanoma patients. Our results support the hypothesis that miRNAs derived from EVs can serve as prognostic or diagnostic liquid biopsy markers in melanoma. We identified EV-derived miRNAs and showed that those miRNAs, which were enriched in melanoma cells and EVs, are also found elevated in serum-derived EVs of patients with metastatic melanoma, but not in healthy subjects.

Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms.

SummaryBackgroundThe prognostic value of tumor-infiltrating lymphocytes (TILs) assessed by machine learning algorithms in melanoma patients has been previously demonstrated but has not been widely adopted in the clinic. We evaluated the prognostic value of objective automated electronic TILs (eTILs) quantification to define a subset of melanoma patients with a low risk of relapse after surgical treatment.MethodsWe analyzed data for 785 patients from 5 independent cohorts from multiple institutions to validate our previous finding that automated TIL score is prognostic in clinically-localized primary melanoma patients. Using serial tissue sections of the Yale TMA-76 melanoma cohort, both immunofluorescence and Hematoxylin-and-Eosin (H&E) staining were performed to understand the molecular characteristics of each TIL phenotype and their associations with survival outcomes.FindingsFive previously-described TIL variables were each significantly associated with overall survival (p<0.0001). Assessing the receiver operating characteristic (ROC) curves by comparing the clinical impact of two models suggests that etTILs (electronic total TILs) (AUC: 0.793, specificity: 0.627, sensitivity: 0.938) outperformed eTILs (AUC: 0.77, specificity: 0.51, sensitivity: 0.938). We also found that the specific molecular subtype of cells representing TILs includes predominantly cells that are CD3+ and CD8+ or CD4+ T cells.InterpretationeTIL% and etTILs scores are robust prognostic markers in patients with primary melanoma and may identify a subgroup of stage II patients at high risk of recurrence who may benefit from adjuvant therapy. We also show the molecular correlates behind these scores. Our data support the need for prospective testing of this algorithm in a clinical trial.FundingThis work was also supported by a sponsored research agreements from Navigate Biopharma and NextCure and by grants from the NIH including the Yale SPORE in in Skin Cancer, P50 CA121974, the Yale SPORE in Lung Cancer, P50 CA196530, NYU SPORE in Skin Cancer P50CA225450 and the Yale Cancer Center Support Grant, P30CA016359.

DNA damage in peripheral blood mononuclear cells in patients with melanoma

Introduction. The incidence and mortality of malignant melanoma have increased steadily over the last decades; therefore, the development of novel diagnostic markers for malignant melanoma is of great importance. The purpose of the study was to assess whether the development of melanoma before any treatment is accompanied by the body changes and, in particular, DNA damage in the mononuclear cells of the peripheral blood of patients. Material and Methods. In 93 patients (26 men and 67 women) admitted to the N.N. Petrov National Medical Research Center of Oncology for surgical treatment of stage T1c-2a-b-3a-b4a-bN0-1 cutaneous malignant melanoma, and in 118 healthy people as a comparison group, the level of damage to DNA in peripheral blood mononuclear cells was studied using the “comet” method. All patients were divided into two groups: group 1 included 45 patients (13 men and 32 women) who were examined before a decision on treatment was made and group 2 consisted of 48 patients (13 men and 35 women) who previously underwent excision biopsy for melanoma. Results. The level of DNA damage in peripheral blood mononuclear cells, assessed by the comet assay, was found to be signifcantly higher in patients with melanoma than in the comparison group. Moreover, the increase in the level of DNA damage was similar both in patients with a primary tumor before starting any treatment and in those who previously underwent excision biopsy for melanoma. The relationship between the level of DNA damage in peripheral blood mononuclear cells and the morphological characteristics of the tumor cells was revealed. The Spearman correlation analysis showed that all parameters that determined DNA damage positively correlated with the thickness of melanoma according to the Breslow’s depth, and the percentage of DNA in the comet and the comet tail moment correlated with the stage of the disease. Conclusion. The development of cutaneous melanoma is accompanied by an increase in the level of DNA damage in peripheral blood mononuclear cells. The level of DNA damage in peripheral blood mononuclear cells refects the changes that occur in the patient’s body under the infuence of the tumor process, which may allow using this indicator as an additional criterion for the diagnosis and aggressiveness of melanoma.

Plasma methylated DNA markers of cutaneous melanoma: Association with PET/CT-positive disease.

9567 Background: Cutaneous melanoma surveillance is important to identify low-volume systemic disease, but imaging is costly and poorly accessible to patients; frequent skin checks lack sensitivity and specificity. We aimed to establish clinical feasibility of a liquid biopsy blood test, which quantifies validated, melanoma-specific, methylated DNA markers (MDMs), previously discovered, and reported by our team, using tissue extracted DNA. Methods: We prospectively collected blood from adult patients with histologically confirmed melanoma metastases and no other internal malignancies (within 5-years) who underwent surveillance by FDG-PET/CT (N = 88). Blood from age- and sex balanced cancer-free controls (N = 100) were compared. From PET/CT, we extracted the number of organs involved, SUV-max, and largest tumor diameter. Unequivocal metastasis was defined as SUV ≥ 4 and largest diameter &gt; 5 mm. Because PET/CT is inadequate for the screening of brain metastases, we excluded the brain from the analysis. MDMs ( chr11.149, HOXA9, chr20.210, FLJ22536, CLIC5, SIX4, chr7.155, chr17.730, chr1.110) were assayed using target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, in blinded fashion. Using a logistic regression approach and nine candidate MDMs, we calculated the sensitivity for detecting patients with metastasis on PET/CT at 100% specificity. Results: 52/88 (59%) of melanoma patients showed evidence of metastasis on PET/CT at the time of blood draw. At 100% specificity, a panel of 4 MDMs ( HOXA9, chr20.210, chr17.730, chr1.110) yielded a sensitivity of 86.5% (45/52 cases) vs. 100 cancer-free controls. When applying this model to the 36 PET/CT-negative patients, specificity was as high as 97.2% (35/36 cases) while maintaining a sensitivity of 86.5% (one patient with a positive test result had a complete metabolic response to binimetinib / encorafenib prior to negative PET/CT). For patients with ≥ 2 organs involved by metastasis, sensitivity was 100% (29/29 cases). False-negative cases had metastasis in single organs and were characterized by minimal tumor burden and oral corticosteroid use. One false-negative patient had localized stage III disease without known primary melanoma. Conclusions: Plasma MDM levels appear highly concordant with FDG-PET/CT in patients with metastatic cutaneous melanoma. A liquid biopsy approach has potential to lower cost and improve patient access to surveillance. Additional prospective studies in larger intended use cohorts are needed to validate our results.

The Genetics of Early-Stage Melanoma in a Veteran Population.

To improve understanding of the genetic signature of early-stage melanomas in Veterans, hotspot mutation profiling using next-generation sequencing (NGS) was performed on melanoma tissue samples from patients at the Iowa City Veterans Affairs Medical Center (VAMC). Genetic analysis identified BRAF (36.3%), TP53 (25.9%), NRAS (19.3%), CDKN2A (11.1%), KIT (8.1%), and BAP1 (7.4%) mutations with the highest prevalence. Although common variants in BRAF were detected at lower rates than what is reported for the general population, 55.6% of cases showed activating mutations in the RAS/RAF pathways. Variants in TP53 and KIT were detected at higher rates than in the general population. Veterans with prior history of melanoma were at significantly higher odds of having TP53 mutation (OR = 2.67, p = 0.04). This suggests that TP53 may be a marker for recurrent melanoma and possibly alternative exposures in the military population. This study provides new information regarding the genetics of melanoma in a Veteran population and early-stage melanomas, highlighting risk factors unique to this population and contributing to the conversation about preventing melanoma deaths in US Military personnel.

EIF6 as a Promising Diagnostic and Prognostic Biomarker for Poorer Survival of Cutaneous Melanoma.

BackgroundSkin cutaneous melanoma (SKCM) is the deadliest skin cancer and has the most rapidly increasing incidences among all cancer types. Previous research elucidated that melanoma can only be successfully treated with surgical abscission in the early stage. Therefore, reliable and specific biomarkers are crucial to melanoma diagnosis since it often looks like nevi in the clinical manifestations. Moreover, identifying key genes contributing to melanoma progression is also highly regarded as a potential strategy for melanoma therapy. In this respect, translation initiator eIF6 has been proved as a pro-tumor factor in several cancers. However, the role of eIF6 in the skin cutaneous melanoma progression and its potential as a prognostic marker is still unexplored.MethodsThe immunochemical analysis of clinical specimens were served to assess eIF6 expression levels. Gene Expression Profiling Interactive Analysis (GEPIA) database consultations allowed us to find the survival rates of the eIF6-overexpressed patients. eIF6 cellular effects were evaluated in an eIF6-overexpressed A375 cell line constructed with a lentivirus. The analysis of down-stream effectors or pathways was conducted using C-Bioportal and STRING databases.ResultsOur results revealed that eIF6 was highly over-expressed in melanomas compared to normal skin specimens, and thus the abnormally high level of eIF6 can be a diagnostic marker for melanoma. The in silica analysis indicated that patients with eIF6 over-expression had lower survival rates than that low-expression in SKCM. Meanwhile, similar results also could be found in the other four types of cancers. In vitro, over-expression of eIF6 increased the proliferation and migration of melanoma cells. Correspondingly, pan-cancer clustering analysis indicated the expression level of intermediate filament proteins was correlated with that of eIF6 expression. In our study, all over-expressed keratin proteins, in accordance with over-expressed eIF6, had a negative correlation with melanoma prognosis. Moreover, the decreased methylation level of keratin genes suggested a new potential regulation mode of eIF6.ConclusionsThe up-regulated eIF6 could be a potential diagnostic and prognostic biomarker of melanoma. This study also provides insights into the potential role of eIF6 in pan-cancer epigenetic regulation.

PRAME Expression in Endometrioid and Serous Endometrial Carcinoma: A Potential Immunotherapeutic Target and Possible Diagnostic Pitfall.

Preferentially expressed antigen in melanoma (PRAME) is a cancer testes antigen initially employed as a diagnostic marker for melanoma. Although negative in most normal tissues, its expression has been reported in benign endometrial glands. Additionally, PRAME expression has been identified in a growing list of solid and hematologic malignancies and is of interest as a predictive biomarker, as cancer vaccination strategies and adoptive T-cell transfer targeting this molecule are under clinical investigation; additionally, PRAME may identify candidates for retinoid therapy. However, expression of PRAME has not been well-studied in endometrial cancers. We herein evaluate PRAME expression in endometrial carcinomas to better characterize its limitations as a diagnostic melanoma marker as well as its potential as a predictive biomarker in endometrial carcinomas. PRAME expression was evaluated in 256 endometrioid (n=235) and serous (n=21) endometrial carcinomas via tissue microarray. In all, 89% (227/256) demonstrated some degree of nuclear PRAME expression, including 88% (207/235) of endometrioid carcinomas and 95% (20/21) of serous carcinomas. Diffuse (>50%) expression was observed in 70% (179/256) of all cases, including 69% (163/235) of endometrioid carcinomas and 76% (16/21) of serous carcinomas. There was no association between degree of expression and grade, mismatch repair protein status, or stage. The widespread expression of PRAME in endometrial carcinomas suggests this marker should not be interpreted as specific for melanoma in this context. However PRAME may have utility as a predictive biomarker in endometrial cancer, and expansion of testing of PRAME-based therapies to endometrioid and serous endometrial carcinomas may lead to new therapeutic options for these endometrial cancer subtypes.

Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma.

Multiplex immunofluorescence staining enables the simultaneous detection of multiple immune markers in a single tissue section, and is a useful tool for the identification of specific cell populations within the tumour microenvironment. However, this technology has rarely been validated against standard clinical immunohistology, which is a barrier for its integration into clinical practice. This study sought to validate and investigate the accuracy, precision and reproducibility of a multiplex immunofluorescence compared with immunohistochemistry (IHC), including tissue staining, imaging and analysis, in characterising the expression of immune and melanoma markers in both the tumour and its microenvironment. Traditional chromogenic IHC, single-plex immunofluorescence and multiplex immunofluorescence were each performed on serial tissue sections of a formalin-fixed paraffin-embedded (FFPE) tissue microarray containing metastatic melanoma specimens from 67 patients. The panel included the immune cell markers CD8, CD68, CD16, the immune checkpoint PD-L1, and melanoma tumour marker SOX10. Slides were stained with the Opal™ 7 colour Kit (Akoya Biosciences) on the intelliPATH autostainer (Biocare Medical) and imaged using the Vectra 3.0.5 microscope. Marker expression was quantified using Halo v.3.2.181 (Indica Labs). Comparison of the IHC and single-plex immunofluorescence revealed highly significant positive correlations between the cell densities of CD8, CD68, CD16, PD-L1 and SOX10 marker positive cells (Spearman’s rho = 0.927 to 0.750, p < 0.0001). Highly significant correlations were also observed for all markers between single-plex immunofluorescence and multiplex immunofluorescence staining (Spearman’s rho >0.9, p < 0.0001). Finally, correlation analysis of the three multiplex replicates revealed a high degree of reproducibility between slides (Spearman’s rho >0.940, p < 0.0001). Together, these data highlight the reliability and validity of multiplex immunofluorescence in accurately profiling the tumour and its associated microenvironment using FFPE metastatic melanoma specimens. This validated multiplex panel can be utilised for research evaluating melanoma and its microenvironment, such as studies performed to predict patient response or resistance to immunotherapies.

PRAME Expression as Helpful Immunohistochemical Marker in Rhabdoid Melanoma.

Background: Rhabdoid melanoma is a rare variant of malignant melanoma with characteristic cytomorphologic features. Due to the potential loss of conventional melanocytic markers, histopathologic diagnosis is often challenging. We hypothesize that immunostaining for PReferentially expressed Antigen in MElanoma (PRAME) might have the potential to uncover the melanocytic origin of these dedifferentiated tumors. Methods: Four cases of rhabdoid primary melanomas were assessed by immunohistochemistry for expression of PRAME and conventional melanocytic markers. Immunohistochemical expression patterns were analyzed in the rhabdoid primaries and, if available, associated metastases. Results: All four cases of rhabdoid primary melanomas showed a strong nuclear positivity for PRAME, while the expression of conventional melanocytic markers S100, MART-1, SOX-10 and HMB-45 was variable between the analyzed cases. Conclusions: In summary, we report four cases of rhabdoid primary melanoma with high to intermediate expression of PRAME despite the partial and variable loss of other melanocytic markers. Hence, PRAME might facilitate the recognition of this highly aggressive entity to avoid misdiagnosis due to histopathologic pitfalls.

Molecular Docking Studies with Garlic Phytochemical Constituents to Inhibit the Human EGFR Protein for Lung Cancer Therapy

In both developed and emerging economies, carcinoma is one of the most frequent malignancies. Non-small cell lung cancer accounts for 80% of all melanoma within India. The basic concept of molecular precision medicine for lung cancer is the EGFR (Epidermal growth factor receptor), which belongs to the tyrosine kinase receptor family (RTKs). Garlic (Allium sativum L), an Indian Traditional spice from the Allium family, appears to be capable of inhibiting cancer cell formation through an apoptotic route. In the present research, the competence and binding ability of various phytochemical compounds from garlic targeting lung melanoma marker protein were assessed. Garlic's medicinal benefits are assumed to be due to key components within the elemental composition, such as organosulfur compounds and flavonoids. There were 19 compounds along with 2 reference compounds that were used for molecular docking purposes. The study revealed that the chemicals investigated are bound effectively to EGFR. When compared to water-soluble and lipid-soluble organosulfur compounds, garlic flavonoids had the anticipated free energy of interaction. Especially in contrast to Alliin, S-Methyl-L-cysteine, Kaempferol, Quercetin, Myrecetin, Apigenin along with Erlotinib demonstrated excellent interaction. Alliin, the much more prevalent component throughout the whole garlic bulb, has a stronger affinity via intermolecular hydrogen bonding, with a binding energy of -7.4 kcal/ Mol. The water-soluble chemical S-Methyl-L-cysteine also had positive interactions with EGFR (-7.3 Kcal/mol). Among the flavonoid groups studied, Quercetin, Myrecetin and Apigenin were found to be the best suitable compound with a binding energy of -8.5 kcal/ Mol. The current findings pave the way for a deeper understanding of garlic's active substance interactions with EGFR. The substances discovered might be utilised as a prototype for anti-cancer treatmentHowever, more research into these compounds' biochemical functions is required before they can be used to treat lung cancer.

NRF3 Decreases during Melanoma Carcinogenesis and Is an Independent Prognostic Marker in Melanoma.

The prognostic significance of the major redox regulator, nuclear factor erythroid-2-related factor 2 (NRF2), is recognized in many cancers, but the role of NRF3 is not studied. Analysis from the Gene Expression Omnibus datasets showed that NRF3 mRNA levels increased from benign to dysplastic naevi (p = 0.04). We characterized the immunohistochemical expression of NRF3 in 81 naevi, 67 primary skin melanomas, and 51 lymph node metastases. The immunohistochemical expression of cytoplasmic NRF3 decreased from benign to dysplastic naevi (p < 0.001) and further to primary melanomas (p < 0.001). High cytoplasmic NRF3 protein expression in pigment cells of the primary melanomas associated with worse melanoma-specific survival in multivariate analysis, specifically in the subgroup of patients with the lymph node metastases at the time of diagnosis (hazard ratio 3.179; 95% confidence interval 1.065-9.493; p = 0.038). Intriguingly, we did not observe associations between NRF3 and the traditional prognostic factors such as Breslow thickness, ulceration, or stage. Together, this data represents the primary description about the role of NRF3 in pigment tumours that is worthy of further explorations.

A proof-of-concept study on the use of a fluorescein-based 18F-tracer for pretargeted PET

BackgroundPretargeted immuno-PET tumor imaging has emerged as a valuable diagnostic strategy that combines the high specificity of antibody-antigen interaction with the high signal and image resolution offered by short-lived PET isotopes, while reducing the irradiation dose caused by traditional 89Zr-labelled antibodies. In this work, we demonstrate proof of concept of a novel ‘two-step’ immuno-PET pretargeting approach, based on bispecific antibodies (bsAbs) engineered to feature dual high-affinity binding activity for a fluorescein-based 18F-PET tracer and tumor markers.ResultsA copper(I)-catalysed click reaction-based radiolabeling protocol was developed for the synthesis of fluorescein-derived molecule [18F]TPF. Binding of [18F]TPF on FITC-bearing bsAbs was confirmed. An in vitro autoradiography assay demonstrated that [18F]TPF could be used for selective imaging of EpCAM-expressing OVCAR3 cells, when pretargeted with EpCAMxFITC bsAb. The versatility of the pretargeting approach was showcased in vitro using a series of fluorescein-binding bsAbs directed at various established cancer-associated targets, including the pan-carcinoma cell surface marker EpCAM, EGFR, melanoma marker MCSP (aka CSPG4), and immune checkpoint PD-L1, offering a range of potential future applications for this pretargeting platform.ConclusionA versatile pretargeting platform for PET imaging, which combines bispecific antibodies and a fluorescein-based 18F-tracer, is presented. It is shown to selectively target EpCAM-expressing cells in vitro and its further evaluation with different bispecific antibodies demonstrates the versatility of the approach.

Disruption of LTBP4 Inhibition-Induced TGFβ1 Activation Promoted Cell Proliferation and Metastasis in Skin Melanoma by Inhibiting the Activation of the Hippo-YAP1 Signaling Pathway.

Melanoma is a malignant tumor derived from melanocytes, which is the most fatal skin cancer. The present study aimed to explore and elucidate the candidate genes in melanoma and its underlying molecular mechanism. A total of 1,156 differentially expressed genes were obtained from the GSE46517 dataset of Gene Expression Omnibus database using the package “limma” in R. Based on two algorithms (LASSO and SVM-RFE), we obtained three candidate DEGs (LTBP4, CDHR1, and MARCKSL1). Among them, LTBP4 was identified as a diagnostic marker of melanoma (AUC = 0.985). Down-regulation of LTBP4 expression was identified in melanoma tissues and cells, which predicted poor prognosis of patients with melanoma. Cox analysis results discovered that LTBP4 with low expression was an independent prognostic factor for overall survival in patients with melanoma. LTBP4 inhibition reduced cell apoptosis and promoted cell proliferation and metastasis. These changes were correlated with the expression levels of caspase-3, Ki67 and E-cadherin. Further, as indicated by tumor formation study of nude mice, LTBP4 silencing improved the tumorigenic ability of melanoma cells. Knockdown of LTBP4 increased the percentage of active TGFβ1 secreted by melanoma cells. CTGF, Gyr61, and Birc5 expression levels were reduced, YAP1 phosphorylation was inhibited, and YAP1 was translocated from the cytoplasm to the nucleus in melanoma cells treated with TGF-β1. These effects were reversed by LTBP4 overexpression. As evidenced by immunofluorescent staining, Western blotting and luciferase reporter assay, LTBP4 overexpression activated the Hippo signaling pathway, which was characterized by the increased nuclear-cytoplasmic translocation of YAP1 and the enhanced phosphorylation of YAP1, MST1, and MOB1. In addition, the effects of LTBP4 overexpression on inhibiting CTGF, Cyr61 and Birc5 expression, promoting the apoptosis, and inhibiting the metastasis and proliferation of melanoma cells were reversed by the overexpression of YAP1 or MST1. In conclusion, the LTBP4-TGFβ1-Hippo-YAP1 axis is a critical pathway for the progression of skin melanoma.

Accurate Delineation of Mucosal Lesions in Treatment-Planning Computed Tomography Using Iodine Paste Markers for Oral Mucosal Melanoma

This technical report introduces the utility of iodine paste markers using endodontic materials for the accurate contouring of mucosal lesions of oral mucosal melanoma, which are difficult to delineate on imaging during the planning of carbon-ion radiation therapy (CIRT). The patient had a primary oral mucosal melanoma located in the palatal mucosa without palatal or maxillary bone invasion. A dental root canal filling material, which is a calcium hydroxide/iodoform nonhardenable paste, was used as a marker. We first performed treatment-planning computed tomography (CT) without an iodine paste marker for mucosal lesions. Subsequently, we placed an iodine paste marker on the palatal mucosal lesion to accurately delineate the mucosal lesions of the palate. Finally, we obtained a reference CT image with an iodine paste marker. Computed tomography without the marker was fused to the reference CT with markers during treatment planning, and the gross tumor volume was contoured. Thereafter, CIRT was delivered without markers. During CIRT, expected acute mucositis was observed in the area of the planning target volume, including melanosis, in accordance with the dose distribution. The use of iodine paste markers for localized mucosal lesions, which are difficult to delineate on CT and magnetic resonance imaging, may be useful for accurately contouring gross tumor volumes on treatment-planning CT.

Mapping of MeLiM melanoma combining ICP-MS and MALDI-MSI methods

Here we developed a powerful tool for comprehensive data collection and mapping of molecular and elemental signatures in the Melanoma-bearing Libechov Minipig (MeLiM) model. The combination of different mass spectrometric methods allowed for detail investigation of specific melanoma markers and elements and their spatial distribution in tissue sections. MALDI-MSI combined with HPLC-MS/MS analyses resulted in identification of seven specific proteins, S100A12, CD163, MMP-2, galectin-1, tenascin, resistin and PCNA that were presented in the melanoma signatures. Furthermore, the ICP-MS method allowed for spatial detection of zinc, calcium, copper, and iron elements linked with the allocation of the specific binding proteins.

A case of malignant melanoma, a possible primary site in the digit, with systemic metastasis in a mini-Rex.

We report a case of systemic metastasis of malignant melanoma in a mini-Rex (Oryctolagus cuniculus). The animal presented with lameness of the right hind limb, swelling of popliteal lymph node, and a black mass on the first digit. Paralysis of hindlimbs and forelimbs, dysuria, and dysphagia progressed over time, and the rabbit died on day 35 from the first visit. At necropsy, many black lesions were observed in multiple organs including the marrow of most bones. Histopathologically, the tumor cells had highly atypical nuclei of various sizes and an abundant eosinophilic cytoplasm, and some cells contained melanin granules. These cells were positive for PNL2 and S-100, melanoma markers, by immunohistochemistry. This is the first report of malignant melanoma in a mini-Rex with severe malignancy and systemic metastasis including the bone marrow.

A simple electrochemical immunosensor based on a chitosan/reduced graphene oxide nanocomposite for sensitive detection of biomarkers of malignant melanoma.

The sensitive and specific detection of tumor biomarkers is crucial for early diagnosis and treatment of malignant melanoma. Immunoassay with a simple sensing interface and high sensitivity is highly desirable. In this work, a simple electrochemical immunosensor based on a chitosan/reduced graphene oxide (CS–rGO) nanocomposite was developed for sensitive determination of an S-100B protein, a tumor marker of malignant melanoma. CS–rGO nanocomposite were prepared by chemical reduction of graphene oxide in the presence of chitosan and modified on glassy carbon electrode (GCE) to provide a biofriendly, conductive, and easily chemically modified matrix for further immobilization of antibodies. Anti-S-100B antibodies were grafted onto the chitosan molecules to fabricate the immunorecognition interface by a simple glutaraldehyde cross-linking method. Electrochemical determination of S-100B was achieved by measuring the decreased current signal of solution phase electrochemical probes, which originated from the increased steric hindrance and insulation caused by the formation of antigen–antibody complexes at the electrode interface. Due to the good conductivity, high surface area, excellent biocompatibility, and good film-forming ability of CS–rGO, the constructed immunosensor exhibited good stability, high selectivity and sensitivity, a wide dynamic range from 10 fg mL−1 to 1 ng mL−1 and a low limit of detection of 1.9 pg mL−1 (S/N = 3). Moreover, the sensor was also applicable for the sensitive detection of S-100B protein in real human serum samples.