Melanoma Markers Research Articles

Fatty acid receptor GPR120: a novel marker for human melanoma.

The correlation between ultraviolet radiation of the skin and melanoma incidence in humans is well established. Interestingly, epidemiologic data suggest also a correlation to an increased BMI pointing to metabolic trigger factors in melanoma pathogenesis. To substantiate this connection, we studied the expression of G-protein-coupled receptor 120 (GPR120), a receptor sensitive to unsaturated long-chain free fatty acids in melanoma tissues. One-hundred fourteen tissue sections histologically confirmed as nevi (n=32), primary melanoma (n=39), and melanoma metastasis (n=43) were immunohistochemically stained against GPR120. The staining was evaluated by three trained dermatopathologists and independently scored. Compared with nevi, primary melanoma and melanoma metastasis showed significantly higher levels of GPR120 staining. Only three out of 32 nevi showed strong GPR120 expression [median immunoreactivity-scoring system (IRS) score: 1, range: 0-10], whereas in primary melanomas 14 out of 39 were highly GPR120-positive (median IRS score: 7, range: 0-12) and in melanoma metastasis 27 out of 43 were highly GPR120-positive (median IRS score: 9, range: 0-12). GPR120 expression and tumor thickness (mm) show a statistically significant correlation in primary melanoma (P=0.011). Moreover, GPR120-positive staining was found throughout the epidermis and in sebaceous and sweat glands, which is yet not described. This study identified GPR120 as a novel marker for melanoma, indicating that melanoma cells are sensitive to free fatty acids. It is tempting to speculate that pharmacologically interfering with GPR120 signaling might improve melanoma therapy.

Sleep-Disordered Breathing Is Independently Associated With Increased Aggressiveness of Cutaneous Melanoma: A Multicenter Observational Study in 443 Patients

Sleep-disordered breathing (SDB) has been associated with a greater incidence and mortality of cancer, although such findings are inconsistent. However, no large studies are currently available to investigate this association in patients with a specific type of cancer. This study seeks to assess potential relationships between SDB severity and aggressiveness markers of cutaneous melanoma. Four hundred and forty-three patients with a diagnosis of melanoma underwent a sleep study within 6months of diagnosis. General demographics were collected, along with melanoma characteristics and polygraphic parameters consisting of the apnea-hypopnea index (AHI) and indices of both continuous and intermittent night-time oxyhemoglobin desaturation (DI4%). An exploration of independent relationships between SDB and various objective melanoma aggressiveness markers (Breslow index, presence of ulceration, presence of regression, mitotic index, stage of severity, damage to the sentinel lymph, and spreading of the melanoma) was performed. Patients in the upper tertiles of AHI or DI4%were 1.94 (95%CI, 1.14-3.32; P=.022) and 1.93 (95%CI, 1.14-3.26; P= .013) times more likely, respectively, to present with aggressive melanoma (Breslow index > 1mm) than those in the lowest tertiles of these sleep attributes after adjustment for age, sex, tumor location, and BMI. This association was particularly prominent among patients< 56 years of age with Breslow index > 2mm. The presence of the additional markers of aggressiveness was also associated with higher AHI and DI4%values. The severity of SDB was independently associated with greater aggressiveness of cutaneous melanoma, particularly among younger patients.

Abstract 3601: Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells

Abstract Recurrence is the major cause of melanoma death due to cell dissemination from primary/metastatic tumor (CTCs). During asymptomatic periods, these cells reside in bone marrow (bone-marrow resident tumor [BMRT cells]) and remain quiescent. We hypothesized that disseminated tumor cells survive in bone marrow (BM) during these periods and evolve to metastatic potency during disease progression. First, we isolated CTC-enriched, Lin-neg population from clinically advanced melanoma patients and performed biomarker expression and mutational profiling to confirm presence of putative CTCs. Lin-neg CTC population contained unique transcriptomics signature with elevated melanoma markers expression (BAGE, MAGEA1, B4GALNT1, S100A3). Further, downstream IPA analysis demonstrated that an upregulation of transcripts for genes involved in cell survival and pro-development functions with concomitant decrease in cell proliferative and inflammation properties. Second, we implanted Lin-neg population in murine xenograft models and isolated HLA+/ Melan-A+ cells population from blood and BM at endpoint. Expression of human (HLA-ABC) and melanoma markers (Melan-A, S100, NG2, CD146) on BMRTCs and CTCs was confirmed by employing: (a) immunofluorescence staining, (b) single-cell DEPArrayTM and CellSearchTM capture, (c) genomic profiling, and (d) organ-site colonization of CTCs with concomitant to BMRTCs in BM. Third, we discovered distinct transcriptomic signature of BMRTCs vs CTCs by differential gene expression profiling. Subsequent pathway analysis showed top five altered canonical pathways in BMRTCs: protein ubiquitination pathway, EIF2, actin cytoskeleton, systemic lupus erythematosus and hypoxia signaling. Of note, a strikingly elevated expression of PTEN in BMRTCs was detected. Fourth, because PTEN binds to USP7, a key component of protein ubiquitination pathway, we evaluated USP7/PTEN axis in CTC-driven BMRTCs modulated metastatic competency. The use of two clinically approved USP7 inhibitors to study their effects on the proliferative capacity of BMRTCs led to a significant reduction of CTCs at metastatic sites. This study provides critical insights to identify biomarkers of melanoma recurrence during the asymptomatic periods of the disease, fostering application of USP7 inhibitors for innovative melanoma therapies for patients with metastasis undetectable disease and/or yet to develop metastasis. Citation Format: Monika Vishnoi, Debasish Boral, Haowen N. Liu, Marc L. Sprouse, Wei Yin, Michael A. Davies, Isabella C. Glitza Oliva, Dario Marchetti. Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3601.

Altered Nuclear Expression of the Deubiquitylase BAP1 Cannot be Used as a Prognostic Marker for Canine Melanoma

SummaryBRCA1-associated protein-1 (BAP1) is a nuclear localized deubiquitylating enzyme that belongs to the ubiquitin c-terminal hydrolase subfamily. The encoded protein is highly homologous between man and dogs, suggesting a functional significance preserved by evolution. BAP1 has multiple properties, including tumour suppressor activity. Loss of BAP1 function is implicated in the oncogenesis of several types of cancers including uveal, mucosal and some cutaneous melanomas in humans, as well as in mesothelioma. In this study we investigate the significance of BAP1 in canine melanoma. Nuclear BAP1 protein was detected in five canine oral melanoma cell lines using an antibody commonly used for analysis of human tissues. BAP1 loss of function mutations often lead to loss of nuclear BAP1 (nBAP1) expression in humans; this is associated with a poorer prognosis in uveal and mucosal melanoma. Therefore, as a prelude to a study evaluating the prognostic significance of nBAP1 expression in dogs, immunohistochemistry (IHC) was used to assess cases of canine melanoma for nBAP1 expression. In 89 cases where tumour cells were identified by melan-A labelling, 100% of tumour cells were positive for nBAP1 expression, including eight uveal tract and 29 oral mucosal melanomas. This finding indicates that BAP1 IHC cannot be used as a prognostic marker in canine uveal and mucosal melanoma. Moreover, this observation suggests that either BAP1 has a different functional significance in canine melanoma or that loss of BAP1 function is achieved by a different route. This is a novel finding that warrants further investigation to determine the comparative biological relevance.

Abstract 4667: The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma

Abstract S100B belongs to the S100 protein family of Ca2+-binding proteins. Prior studies have shown that S100B is a biological marker for malignant melanoma. Increases in S100B levels are predictive of disease progression, recurrence, and overall low survival in the patients. It was shown previously that S100B binds to p53 and down-regulates its tumor suppression activities including growth arrest and apoptosis. Chemical biology and structure-based drug design methods are underway to inhibit S100B and restore p53 function. However, highly specific S100B inhibitors are required, so S100B can be blocked in melanoma without impairing S100A1 function in skeletal and cardiac muscle. With this goal in mind, a fragment-based screening approach using NMR and computational approaches is being applied. Results to date indicate that these methods can be used to identify specific small molecule fragments that target S100B and not S100A1. The fragments that uniquely bind S100B will then be considered with structural data of existing S100B-SBiX complexes to improve their specificity for binding S100B (i.e. versus S100A1). The inhibitor design strategy will also include improving the stability of the compounds their cell permeability, and their overall drug-like properties. The most promising of these new inhibitors will ultimately be used to target high levels of S100B found in malignant and rescue p53-dependent tumor suppression pathways in vivo with the goal of them having therapeutic value for treating malignant melanoma. Citation Format: Sreya Mukherjee, Shardell Spriggs, Paul Wilder, Wenbo Yu, Kristen Varney, Alexander D. MacKerell, David J. Weber. The use of fragment-based screening approaches to improve the specificity of inhibitors targeting malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4667.

Canine Oral Osteocartilaginous Malignant Amelanotic Melanoma with Pulmonary Metastasis

Background: Melanomas are typically malignant neoplasms commonly observed in the oral cavity of dogs. The classical presentation of melanomas with characteristic melanin pigmentation is easy to diagnose; however, in some cases, the lack of melanin production in the amelanotic oral tumors cause a delay in establishing the precocious diagnosis and consequent treatment. The aim of this report was to evaluate the histopathological and immunohistochemical aspects of an oral amelanotic melanoma with osteocartilaginous formation and metastasis in a dog, in a temporal way.Case: A 10-year-old male German Shepherd dog, presenting mouth bleeding with an amelanotic melanoma located between the upper incisors was received at the Veterinary Hospital of the Catholic University Dom Bosco (UCDB), Campo Grande, MS, Brazil. The animal was clinically evaluated and radiography was performed. The tumor was surgically removed and a sample was collected for histopathological examination that revealed spindloid and some epithelioid morphological cell types surrounded by a fibromatous matrix with moderate amounts of fibrovascular stroma. Approximately 1 month after surgical removal, recovery of the tumor was observed, and a second clinical analysis and collection of sample were performed. These procedures were repeated three times showing the same histopathological characteristics added by myxoid, chondroid, fibromatous tissue, and small groups of chondrocytes as well as central areas of irregular mineralized spicules. X-ray examination revealed proliferative and lytic bone infiltration in the jaw. Immunohistochemical analysis for melanocytic differentiation markers was performed showing positivity to Melan-A, tyrosinase and HMB-45 immunoreactivity, while no S100 reactivity was detected. After 11 months of the first biopsy, pleural effusion and radiopaque disseminated nodules of 1cm in the lungs were detected by X-ray. The animal died and necropsy was conducted. Multiple masses were observed in the lung and at the parietal pleura, suggesting lung metastasis by the positivity for Melan-A.Discussion: The dog was first diagnosed with fibromatous epulis based on the observation of fibroblastic tissue and spindle cells with intense vascularization associated to the site of the tumor and its macroscopic aspect. In the subsequent follow-ups the tumor displayed malignant characteristics observed by recurrence after each surgery, as the tumor returned even larger, aggressive, and infiltrative. From the second biopsy, the histopathological analysis showed the undifferentiated character of epithelioid neoplastic cells, demonstrated by the increase of cartilage and osteoid tissue and the mineral deposit. The phenomenon of the tumor stroma to form cartilage and bone is highlighted here because myxoid change and cartilage formation were frequently observed at the site where amelanotic spindle cells were actively proliferating. It is possible that neoplastic melanocytic cells themselves were involved in the development of the osteocartilaginous areas. Although no cytoplasmic melanin pigmentation was found in the tumor fragments, specific melanocytic markers for melanoma detected neoplastic melanocytes and unmelanized melanosomes. The positive reaction for Melan-A, HMB-45, and tyrosinase in the epithelioid, spindle, and cartilaginous cell groups of the neoplasia indicated amelanotic melanoma with osteocartilaginous differentiation. The negative HMB-45, S100, and tyrosinase expression in lung metastasis may be due to the fact that melanomas express aberrant markers and are also known to display occasional loss of their classic immunophenotype. Amelanotic melanoma can be underdiagnosed due to rapid progression of the tumor allied to the dedifferentiation ability of melanocytes. Thus, the follow up study of cell morphology and immunohistochemical analysis for melanogenic factors can be important determinants in diagnosis.

PD-L1 expression by tumor cell lines: A predictive marker in melanoma.

Prognostic biomarkers for patients with melanoma after lymph node resection are of clinical relevance and could thus enable the identification of patients who therefore would most benefit from adjuvant treatment. The aim of this work was to determine, using an in vitro model, whether immune-related biomarkers, such as MHC-class I and II, melanoma-associated antigens, IDO1 and PD-L1, could also be relevant to predict the risk of relapse of patients with stage III melanoma after lymph node resection. We established tumor cell lines from metastatic lymph nodes of 50 patients with melanoma. The expression of investigated biomarkers was determined on untreated and IFN-γ treated melanoma cell lines using flow cytometry. Among the selected biomarkers, the IFN-γ-induced expression of PD-L1 and IDO1 was associated with an increased risk of relapse (P=.0001 and P=.013, respectively) and was also associated with death for IDO1 (P=.0005). In the future, this immunologic signature could permit the identification of patients at higher risk of relapse and justifying an adjuvant treatment using immunotherapy.

Graphene-Augmented Nanofiber Scaffolds Trigger Gene Expression Switching of Four Cancer Cell Types.

Three-dimensional (3D) customized scaffolds are anticipated to provide new frontiers in cell manipulation and advanced therapy methods. Here, we demonstrate the application of hybrid 3D porous scaffolds, representing networks of highly aligned self-assembled ceramic nanofibers, for culturing four types of cancer cells. Ultrahigh aspect ratio (∼107) of graphene augmented fibers of tailored nanotopology is shown as an alternative tool to substantially affect cancerous gene expression, eventually due to differences in local biomechanical features of the cell–matrix interactions. Here, we report a clear selective up- and down-regulation of groups of markers for breast cancer (MDA-MB231), colorectal cancer (CaCO2), melanoma (WM239A), and neuroblastoma (Kelly) depending on only fiber orientation and morphology without application of any other stimulus. Changes in gene expression are also revealed for Mitomycin C treatment of MDA-MB231, making the scaffold a suitable platform for testing of anticancer agents. This allows an opportunity for selective “clean” guidance to a deep understanding of mechanisms of cancer cells progressive growth and tumor formation without possible side effects by manipulation with the specific markers.

1243 High Rab27a expression is associated with poor melanoma prognosis and promotes melanoma cell invasion and metastasis via regulation of pro-invasive exosomes

Despite recent advances in targeted and immune-based therapies, metastatic melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis are critical to identify new therapeutic strategies and improve treatment strategies and patient outcomes. Here, using clinical samples, we identified that Rab27a was overexpressed in a subset of melanomas, which correlated with poor survival. To further dissect the function of Rab27a in melanoma, we performed CRISPR-knockout and shRNA-knockdown of this gene in melanoma cell lines. We demonstrated that Rab27a loss inhibited proliferation, colony formation, 3D spheroid invasion and motility in vitro, as well as spontaneous metastasis in vivo. The reduced invasion phenotype observed with Rab27a knockdown was rescued by Rab27a-replete exosomes, indicating that exosomes drive Rab27a-mediated invasion. Further studies revealed that while Rab27a loss did not alter the number of exosomes secreted, there were changes in exosome morphology and the abundance of exosomal proteins associated with cancer cell migration and metastasis. These findings expand on a growing body of literature indicating important roles for Rab27a in regulating cancer cell biology, and further highlights its potential to be utilized as a clinical prognostic marker or therapeutic target in melanoma.

T cell-induced CSF1 promotes melanoma resistance to PD1 blockade.

Colony-stimulating factor 1 (CSF1) is a key regulator of monocyte/macrophage differentiation that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). We show that CSF1 is expressed in human melanoma, and patients with metastatic melanoma have increased CSF1 in blood compared to healthy subjects. In tumors, CSF1 expression correlated with the abundance of CD8+ T cells and CD163+ TAMs. Human melanoma cell lines consistently produced CSF1 after exposure to melanoma-specific CD8+ T cells or T cell-derived cytokines in vitro, reflecting a broadly conserved mechanism of CSF1 induction by activated CD8+ T cells. Mining of publicly available transcriptomic data sets suggested co-enrichment of CD8+ T cells with CSF1 or various TAM-specific markers in human melanoma, which was associated with nonresponsiveness to programmed cell death protein 1 (PD1) checkpoint blockade in a smaller patient cohort. Combination of anti-PD1 and anti-CSF1 receptor (CSF1R) antibodies induced the regression of BRAFV600E -driven, transplant mouse melanomas, a result that was dependent on the effective elimination of TAMs. Collectively, these data implicate CSF1 induction as a CD8+ T cell-dependent adaptive resistance mechanism and show that simultaneous CSF1R targeting may be beneficial in melanomas refractory to immune checkpoint blockade and, possibly, other T cell-based therapies.

Correlation of Immunocytochemistry of BRCA1-associated Protein-1 (BAP1) With Other Prognostic Markers in Uveal Melanoma

Prior studies have shown that nuclear reactivity for BRCA1-associated protein-1 (BAP1) yields prognostic information for paraffin-embedded uveal melanomas. Lacking are immunocytochemical studies of BAP1 on fine needle aspiration biopsies of uveal melanoma that correlate with prognosis or other markers of prognosis. Our purpose was to fill this gap. Experimental laboratory study. Fine needle aspiration biopsies were performed prospectively on 113 patients with uveal melanomas, garnering limited subsets of cases for comparison. Agreement between immunocytochemistry for BAP1 nuclear staining vs chromosome 3 ploidy analysis and gene expression profiling was assessed by 2× 2 contingency table analysis. The presence or absence of suppression of nuclear expression of BAP1 was strongly associated (73%, P= .000002) with monosomy and disomy chromosome 3, respectively. BAP1 nuclear expression was also correlated with gene expression profiling. Chromosome 3 ploidy analysis correlated with gene expression profiles. When adequate material is obtained, immunocytology using BAP1 is a potentially informative tool for prognostication of uveal melanoma.

Hu antigen R (HuR) heterogeneous expression quantification as a prognostic marker of melanoma.

Prognostic markers for melanoma, particularly for stage II disease, are needed for the risk-benefit evaluation of future adjuvant therapies. The mainly nuclear RNA-binding protein human antigen R (HuR) regulates the protein expression of thousands of mRNAs, its own heterogeneous expression could therefore reflect tumor heterogeneity and plasticity. Here, we evaluate its quantification in primary melanoma as a marker of metastatic outcome. We conducted an immunohistochemistry-based automated quantification of HuR nuclear expression heterogeneity in primary melanomas, most with Breslow thickness ≥ 1 mm and calculated the dimensionless fourth moment, that is, the kurtosis of HuR (HuR K) expression distribution. Twelve tumors from patients with no metastatic disease were compared to a similar number of tumors from patients who had metastatic disease at 2 years follow up. HuR K value appeared significantly higher in the non-metastatic group comparatively to the metastatic group (P = 2.84 × 10-3 , 1-tailed Wilcoxon rank-sum test). Moreover, compared to the Breslow thickness, HuR K value appeared as a more robust marker of metastatic outcome (respective areas under receiver operating characteristic curves 0.84 and 0.87). Our data need confirmation on a large cohort, however strongly suggest that HuR expression heterogeneity quantification using kurtosis, could be used as a prognostic marker in melanoma.

PHLPP1 mediates melanoma metastasis suppression through repressing AKT2 activation.

PI3K/AKT pathway activation is thought to be a driving force in metastatic melanomas. Members of the pleckstrin homology (PH) domain leucine-rich repeat protein Ser/Thr specific phosphatase family (PHLPP1 and PHLPP2) can regulate AKT activation. By dephosphorylating specific serine residues in the hydrophobic motif, PHLPP1 and PHLPP2 restrain AKT signalings, thereby regulating cell proliferation and survival. We here show that PHLPP1 expression was significantly downregulated or lost and correlated with metastatic potential in melanoma. Forcing expression of either PHLPP1 or PHLPP2 in melanoma cells inhibited cell proliferation, migration, and colony formation in soft agar; but PHLPP1 had the most profound inhibitory effect on metastasis. Moreover, expression of PH mutant forms of PHLPP1 continued to inhibit metastasis, whereas a phosphatase-dead C-terminal mutant did not. The introduction of activated PHLPP1-specific targets AKT2 or AKT3 also promoted melanoma metastasis, while the non-PHLPP1 target AKT1 did not. AKT2 and AKT3 could even rescue the PHLPP1-mediated inhibition of metastasis. An AKT inhibitor blocked the activity of AKT2 and inhibited AKT2-mediated tumor growth and metastasis in a preclinical mouse model. Our data demonstrate that PHLPP1 functions as a metastasis suppressor through its phosphatase activity, and suggest that PHLPP1 represents a novel diagnostic and therapeutic marker for metastatic melanoma.

Nestin expression in primary and metastatic uveal melanoma - possible biomarker for high-risk uveal melanoma.

Nestin, a member of the intermediate filament protein family, has been described as a putative cancer stem cell marker (CSC) in uveal melanoma and poor prognostic factor in a variety of tumours, including cutaneous melanoma. In this study, we examined the expression of nestin in primary (PUM) and metastatic uveal melanoma (MUM) samples, and correlated the findings with histological, clinical and survival data. Nestin expression was assessed by immunohistochemistry in 141 PUM and 26 MUM samples; 11 PUM cases were matched with their corresponding metastases. The percentage of tumour cells expressing nestin was scored by three independent observers. Statistical analysis of all data was performed with SPSS. Nestin expression was identified in both the cytoplasm and membrane of UM cells. Increased expression of nestin in PUM samples was associated with known poor prognostic parameters, including epithelioid cell morphology (p<0.001), closed loops (p=0.001), higher mitotic count (p<0.001), monosomy 3 (p=0.007) and chromosome 8q gain (p<0.001). Primary uveal melanoma (PUM) with nestin expression levels above a cut-off value of 10% [as determined by receiver operating characteristic (ROC) analysis] was associated with a significantly reduced survival time (Log-rank, p=0.002). In MUM, a higher percentage of nestin-positive tumour cells combined with poor prognostic markers in the PUM led to a shorter survival time following the development of metastases. In conclusion, increased nestin expression in PUM is a predictor of a tumour phenotype associated with metastatic progression and reduced survival time at onset of metastasis.

118 High-Grade Angiosarcoma Masquerading As A Simple Cyst and Histiocytic/Dendritic Cell Neoplasm With Histiocyte-Like Cells—A Diagnostic Conundrum

High-grade, poorly differentiated angiosarcoma is a rare, aggressive malignant neoplasm, comprising 1%-2% of head and neck sarcomas. Diagnosis is often delayed, due to its putatively innocuous clinical appearance combined with the unusual morphology. We report here a case of high-grade angiosarcoma in an elderly patient mimicking clinically and radiologically a benign cyst, and histiocyte-like cells on the morphology. The specimen was fixed in 10% buffered formalin solution and embedded in paraffin using routine procedures. Dewaxed paraffin sections were stained with hematoxylin and eosin. Immunoperoxidase stains were performed on the deparaffinized sections using an avidin-biotin-peroxidase complex. The surgical biopsy revealed histiocytic-like cells characterized by medium to large round nuclei, vesicular chromatin, small nucleoli, abundant eosinophilic cytoplasm, and ill-defined cell borders. These cells are intermixed with chronic inflammatory cells and surrounded by the peripheral cuff of lymphoid cells. Rare multinucleated and multilobulated cells were also identified. No vasoformative structures were identified. On the basis of histomorphology alone, a follicular dendritic sarcoma or other histiocytic sarcoma was considered; however, immunohistochemical stains (CD35, CD23, CD21) were negative in the malignant population, as were the melanoma markers (S100, HMB-45, SOX10, and Melan A) and epithelial markers (EMA, AE1/AE3, CK5/6, CK8/18, and p40); B- and T-cell markers stained only admixed mature lymphocytes. In contrast to these negative results, immunohistochemical studies provided compelling evidence of endothelial differentiation showing positive expression of numerous vascular markers, including CD31, CD34, D2-40, ERG, and factor VIII. A diagnosis of poorly differentiated angiosarcoma was rendered. High-grade, poorly differentiated angiosarcoma has dismal prognosis with high risk of recurrences. Knowledge of its atypical clinical and radiological presentation, as well as awareness of its unusual morphology of histiocyte-like cells can lead to the early diagnosis and prompt treatment.

Genetic polymorphisms may influence the vertical growth rate of melanoma.

Background: Identification of new predictive markers in melanoma is of great clinical importance. This study was aimed to analyze association between selected common variants in the cancer susceptibility genes and melanoma progression at the time of diagnosis.Material and Method: The study included 243 consecutive patients with melanoma. Genotyping was performed using real-time PCR.Results: Our data revealed modest association between xeroderma pigmentosum complementation group D (XPD) codon 312 polymorphism and tumor thickness (as defined by Breslow score; XPD D312N CC: 3.00 ± 3.78mm, CT: 1.71 ± 2.48mm, TT: 2,53 ± 3,24mm, P=0.023). The CT genotype in XPD D312N polymorphism was more frequently represented in non-invasive melanomas compared to deeply penetrating tumors. None of the common SNPs in cyclin dependent kinase inhibitor 2A (CDKN2A), vitamin D receptor (VDR), melanocortin 1 receptor (MC1R) were associated with Breslow depth.Conclusion: These findings suggest that genetic alteration in XPD contributes to melanoma progression and may be a potential diagnostic and molecular prognostic marker.

WIPI1, BAG1, and PEX3 Autophagy-Related Genes Are Relevant Melanoma Markers.

ROS and oxidative stress may promote autophagy; on the other hand, autophagy may help reduce oxidative damages. According to the known interplay of ROS, autophagy, and melanoma onset, we hypothesized that autophagy-related genes (ARGs) may represent useful melanoma biomarkers. We therefore analyzed the gene and protein expression of 222 ARGs in human melanoma samples, from 5 independent expression databases (overall 572 patients). Gene expression was first evaluated in the GEO database. Forty-two genes showed extremely high ability to discriminate melanoma from nevi (63 samples) according to ROC (AUC ≥ 0.85) and Mann-Whitney (p < 0.0001) analyses. The 9 genes never related to melanoma before were then in silico validated in the IST online database. BAG1, CHMP2B, PEX3, and WIPI1 confirmed a strong differential gene expression, in 355 samples. A second-round validation performed on the Human Protein Atlas database showed strong differential protein expression for BAG1, PEX3, and WIPI1 in melanoma vs control samples, according to the image analysis of 80 human histological sections. WIPI1 gene expression also showed a significant prognostic value (p < 0.0001) according to 102 melanoma patients' survival data. We finally addressed in Oncomine database whether WIPI1 overexpression is melanoma-specific. Within more than 20 cancer types, the most relevant WIPI1 expression change (p = 0.00002; fold change = 3.1) was observed in melanoma. Molecular/functional relationships of the investigated molecules with melanoma and their molecular/functional network were analyzed via Chilibot software, STRING analysis, and gene ontology enrichment analysis. We conclude that WIPI1 (AUC = 0.99), BAG1 (AUC = 1), and PEX3 (AUC = 0.93) are relevant novel melanoma markers at both gene and protein levels.

Abstract B59: Role of Dicer on human melanoma progression and resistance

Abstract Melanoma is a serious public health problem that affects the population of Latin America and is increasing rapidly compared to other types of tumors. The growth or change in this type of tumor is progressive and is in the horizontal and/or vertical direction. Breslow thickness (vertical staging) is the tumor thickness in the dermis where patients with lesion thickness less than 0.75 mm have good prognosis, unlike lesion thickness larger than 3 mm. This stage is classified into T (primary tumor): T0 (with no evidence of primary tumor); Tis (in situ melanoma); T1 (1 mm); T2 (1.01 to 2 mm); T3 (2.01 to 4 mm); and T4 (above 4 mm). Knowing some molecular markers of melanoma is essential for the identification of genetically predisposed individuals, as well as early detection of the disease. Recent works has demonstrated that microRNAs may be involved in modulation of melanoma. Dicer is an essential member of the RNase III family which controls maturation of miRNAs in the cytoplasm from microRNA precursors (pre-miRNAs). The upregulation of Dicer is associated with aggressive features and proliferation of melanoma and no other skin tumors such as carcinomas and sarcoma. In our present study, we found that the knockdown expression of Dicer using the CRISPR/CAS9 in melanoma cell line induced more chemosensitivity to cisplatin and these changes correlated with the downregulation of Dicer. We examined the consequence of Dicer knockdown on melanoma cell proliferation, clonogenic assays, and overall survival. We also compared Dicer in tissue from patients diagnosed with melanoma in different stages with the prognosis of these patients, suggesting that the reduction in Dicer expression is associated with melanoma progression. Considering the fundamental and multiple biologic roles of Dicer in various cellular processes, our results suggest that modulation of Dicer in melanoma should become a promising therapeutic candidate for further clinical application. Note: This abstract was not presented at the conference. Citation Format: Nathalia Cruz de Victo, Sandy Adjemian, Romulo dos Santos Sobreira Nunes, Rildo Aparecido Volpini, Mara Huffenbaecher Giavina-Bianchi, Cyro Festa Neto, Niels Olsen Saraiva Câmara, Gustavo Pessini Amarante-Mendes, Jacqueline de Fátima Jacysyn. Role of Dicer on human melanoma progression and resistance [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B59.

Prospective Validation of Molecular Prognostic Markers in Cutaneous Melanoma: A Correlative Analysis of E1690.

Purpose: To validate the prognostic impact of combined expression levels of three markers (SPP1, RGS1, and NCOA3) in melanoma specimens from patients enrolled in the E1690 clinical trial of high-dose or low-dose IFNα-2b versus observation.Experimental Design: Tissue was available from 248 patients. Marker expression was determined by digital imaging of immunohistochemically stained slides. The prognostic impact of each marker was first assessed by recording its expression value relative to the median. A multimarker index was then developed to combine marker expression levels by counting for each patient the number of markers with high expression. The impact of the multimarker index on relapse-free survival (RFS) and overall survival (OS) was assessed using Kaplan-Meier analysis, and both univariate and multivariate Cox regression analyses.Results: By Kaplan-Meier analysis, high multimarker expression scores were significantly predictive of RFS (P < 0.001) and OS (P < 0.001). Stepwise multivariate Cox regression analysis with backward elimination that included routine clinical and histologic prognostic factors revealed high multimarker expression scores and tumor thickness as the only factors significantly and independently predicting RFS and OS. Stepwise multivariate Cox regression analyses that also included treatment type and number of positive lymph nodes generated identical results for both RFS and OS. In the molecularly defined low-risk subgroup, patients treated with high-dose IFN had a significantly improved RFS compared with patients in the other two subgroups (P < 0.05).Conclusions: These results validate the independent impact of combined expression levels of SPP1, RGS1, and NCOA3 on survival of melanoma in a prospectively collected cohort. Clin Cancer Res; 23(22); 6888-92. ©2017 AACR.

Ablation of epidermal RXR\u03b1 in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas

BackgroundUnderstanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation.MethodsWe generated a trigenic mouse model system (RXRαep−/−| Tyr-NRASQ61K | CDK4R24C/R24C) harboring an epidermal knockout of Retinoid X Receptor α (RXRαep−/−), combined with oncogenic NRASQ61K (constitutively active RAS) and activated CDK4R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRαep−/− mice compared to the control mice with functional RXRα.ResultsHere we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRαep−/− mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRαep−/− mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRαep−/− mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRASQ61K and CDK4R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα.ConclusionsOur study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4R24C/R24C and oncogenic NRASQ61K.