Purpose: NRAS and BRAF mutations are common in cutaneous melanomas, although rarely detected mutually in the same tumor. Distinct clinical correlates of these mutations have not been described, despite in vitro data suggesting enhanced oncogenic effects. This study was designed to test the hypothesis that primary human cutaneous melanomas harboring mutations in NRAS or BRAF display a more aggressive clinical phenotype than tumors wild type at both loci.Experimental Design: Microdissection of 223 primary melanomas was carried out, followed by determination of the NRAS and BRAF mutational status. Genotypic findings were correlated with features known to influence tumor behavior including age, gender, Breslow depth, Clark level, mitotic rate, the presence of ulceration, and American Joint Committee on Cancer (AJCC) staging.Results: Breslow depth and Clark level varied significantly among the genotypes, with NRAS mutants showing the deepest levels and wild-type tumors the least depth. Ulceration also differed significantly among the genotypes, with BRAF mutants demonstrating the highest rate. In addition, tumors with mutated NRAS were more likely to be located on the extremities. Patients whose tumors carried either mutation presented with more advanced AJCC stages compared with patients with wild-type tumors, and specifically, were more likely to have stage III disease at diagnosis. Overall survival did not differ among the 3 groups.Conclusions: Distinct clinical phenotypes exist for melanomas bearing NRAS and BRAF mutations, whether considered together or separately, and are associated with features known to predict aggressive tumor behavior. The impact of these mutations is most evident at earlier stages of disease progression.

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A lack of consensus exists regarding the relative rates of NRAS and BRAF mutations in the radial (RGP) and vertical (VGP) growth phases of individual melanoma tumors. This study was conducted to test the hypothesis that mutations are acquired with progression from RGP to VGP. Using laser capture microdissection, pure tumor DNA was obtained from fifteen in-situ melanomas, and from the RGP and VGP of twenty-nine invasive tumors. NRAS exon 2 and BRAF exon 15 DNA were amplified by PCR and sequenced. Mutations were present in six of fifteen in-situ melanomas (40%). Sixteen of twenty-nine invasive tumors exhibited RGP mutations (55.2%); 22 showed VGP mutations (75.9%). Paired RGP/VGP mutation analysis revealed a trend toward discordance in the distribution of mutations, favoring VGP localization (p = 0.07). Twelve of fifteen samples with mutations in both phases had an increased proportion of mutated DNA in the VGP, measured on DNA chromatograms (p = 0.08). Limitations of this study include a relatively small sample cohort selected for technical reasons from a larger population, presenting the risk of selection bias. These concerns notwithstanding, our findings support the hypothesis that NRAS and BRAF mutations increase with tumor progression from superficial to invasive disease.

Cutaneous melanomas can be divided into three mutually exclusive genetic subsets: tumors with mutated BRAF, tumors with mutated NRAS, and tumors wild type at both loci (wt/wt). Targeted therapy for melanoma has been advancing with agents directed to mutated BRAF, accounting for 50% of melanoma patients. The c-Met pathway is known to play a role in melanoma tumorigenesis and preliminary data from our laboratory suggested that this pathway is preferentially activated in NRAS-mutated tumors. The objective of this study was to test the hypothesis that melanomas carrying the mutated NRAS genotype are uniquely sensitively to c-Met inhibition, thus providing rationale for therapeutic targeting of c-Met in this patient cohort. Using primary human melanomas with known BRAF/NRAS genotypes, we observed greater immunostaining for phosphorylated (activated) c-Met in NRAS-mutated and wt/wt tumors, compared to BRAF-mutated tumors. NRAS-mutated and wt/wt cell lines also demonstrated more robust c-Met activation in response to hepatocyte growth factor (HGF). Knock-down of mutated N-Ras, but not wild type N-Ras, by RNA interference resulted in decreased c-Met phosphorylation. Compared to BRAF mutants, NRAS-mutated melanoma cells were more sensitive to pharmacologic c-Met inhibition in terms of c-Met activation, Akt phosphorylation, tumor cell proliferation, migration, and apoptosis. This enhanced sensitivity was observed in wt/wt cells as well, but was a less consistent finding. Based on these experimental results, we propose that c-Met inhibition may be a useful therapeutic strategy for melanomas with NRAS mutations, as well as some tumors with a wt/wt genotype.

Background Oral melanoma (OM) in dogs is an aggressive malignancy, with clinical behavior resembling cutaneous melanomas in humans. Melanoma in humans is promoted by an inflammatory environment which is contributed to by leptin and inducible nitric oxide synthase (iNOS). Objective To determine if the patterns of leptin and iNOS expression are similar in OM in dogs and cutaneous melanomas in humans. Animals Twenty client-owned dogs. Methods Retrospective case study. Immunostaining of the OM tumors from each dog was scored for percentage and intensity of leptin and iNOS expression. Mitotic index was used as an indicator of tumor aggression. Results Leptin was detected in ≥ 75% of the tumor cells in specimens from eleven dogs. One tumor expressed leptin in ≤ 25% of the cells. The intensity of leptin expression was variable with six, nine, and five cases exhibiting low, moderate, and high intensity staining, respectively. OM with the lowest percentage of iNOS positive cells displayed the highest mitotic indices (p = 0.006, ANOVA) Conclusions and Clinical Importance The expression of leptin is a common finding in melanomas in dogs. These data suggest the possibility of future clinical applications, such as measuring the concentrations of plasma leptin as a screening tool or leptin as a target for therapy. The relevance of iNOS is not as clear in dogs with OM, for which other directed therapeutics might be more appropriate.

Melanoma is highly heterogeneous in vivo compared to two-dimensional (2D) monolayer adherent cells in culture. Using human A375 and WM793 mouse xenografts, we consistently observed a significant increase of HIF-1α expression in many live tumor cells by Immunohistochemistry. This distinct feature confirms that some melanoma cells exist under hypoxia in vivo. We hypothesize that melanoma cells survive under hypoxic conditions due to unique growth and survival pathways, which are likely to alter their sensitivity to current therapeutics. We employed a three-dimensional (3D) culture system with an inorganic nanoscale scaffolding (SCIVAX NanoCulture Plate, NCP), to mimic part of the in vivo system. Human B-RAF(V600E) mutated melanoma cell lines, 451Lu, A375, and MEL1617, were confirmed to form spheroids with NCP in standard RPMI medium with 5% FBS under normoxic incubator conditions. The majority of melanoma cells in spheroids were identified as hypoxia as indicated by the hypoxic probe LOX-1. The fluorescence of LOX-1 is quenched by oxygen and increased in response to low levels of oxygen, which was consistently negative in parallel monolayer cultures of these same three melanoma cell lines. Furthermore, cells within the melanoma spheroids differentially expressed HIF-1α and VEGF, at both mRNA and protein levels; also both were undetectable in monolayer cultures in all three tested cell lines. Using this system, melanoma spheroids and 2D-monolayer cultures were treated with the B-RAF(V600E) inhibitor, Vemurafenib. Then the cell viability and expression of signaling markers were compared between 2D and 3D cultures. Data from our MTT assays indicated that the 3D melanoma spheroids were more resistant to Vemurafenib than 2D-cultured cells by 20-30% in all three tested cell lines. We then employed a human phospho-kinase array (R&D System) to study multiple kinase pathways related to hypoxia, and found that the phosphorylated c-Met was significantly increased in 3D melanoma spheroids than 2D-cultured cells, and was confirmed by western blot. This data suggested that hypoxia drove c-Met activation in melanoma spheroids. By employing preclinical c-Met inhibitor in 3D culture, we found that it significantly prevent 3D-spheroid formation (40∼60%) and HIF-1α expression. Combined, evidence suggested that hypoxia drove c-Met activation and resistance to Vemurafenib in melanoma cells of 3D spheroids, which may partially reflect the cellular signature of melanoma cells under hypoxia in vivo. Taking advantage of the ability of c-Met inhibitor preventing melanoma spheroid formation, we proposed that combination of c-Met inhibitor and Vemurafenib was a potential therapy strategy to overcome resistant melanoma cells under hypoxia in patients. Further experiments on melanoma spheroids and mouse model to address this concept are in progress. Citation Format: Yong Qin, Victoria R. Greene, Chandrani Chattopadhyay, Suhendan Ekmekcioglu, Chengwen Liu, Elizabeth A. Grimm. Induction of hypoxia in 3D human melanoma spheroids leads to c-Met activation and resistance to Vemurafenib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2013-2936

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