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Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

Patients with metastatic melanoma have historically had dismal outcomes. The last several years has seen the emergence of effective immune and targeted therapies for metastatic melanoma. Targeted therapies have primarily impacted the 40-50% of patients with BRAF(V600) mutated melanoma. The remainder of patients with advanced melanoma harbor a wide spectrum of mutations other than BRAF(V600) that are associated with unique pathophysiological, prognostic, and therapeutic implications. The treatment of this subset of patients is a challenging problem. In recent years, preclinical and early clinical studies have suggested that inhibitors of mitogen activated protein kinase (MAPK) pathway and parallel signaling networks may have activity in treatment of BRAF(V600) wild-type (WT) melanoma. In this review, we will discuss available and developing therapies for BRAF WT patients with metastatic melanoma, particularly focusing on molecular targeted options for various genetically defined melanoma subsets.

Background: Ipilimumab improves overall survival (OS) in advanced melanoma. Acral melanoma is an uncommon clinical subtype of this disease associated with poor prognosis. The clinical activity of ipilimumab has not been well-defined in advanced acral melanoma., Methods: We retrospectively reviewed the demographics, treatment history, and clinical outcomes for all patients with acral melanoma treated with ipilimumab from two academic centers between February 2006 and June 2013. Using Cox proportional hazards models, we assessed for factors that correlated with OS., Results: A total of 35 patients with acral melanoma received ipilimumab. Melanomas arose on volar surfaces (n = 28) and subungual sites (n = 7); stage M1c disease was present in 54%, and 45% had elevated serum lactate dehydrogenase (LDH). Best response by RECIST 1.1 criteria was complete response in 1 patient, partial response in 3, and stable disease (SD) in 4 for an objective response rate (ORR) of 11.4% and a clinical benefit rate (ORR + SD) at 24 weeks of 22.9%. Median progression-free survival was 2.5 months (95% confidence interval [CI]: 2.3-2.7 months); median OS was 16.7 months (95% CI: 10.9-22.5 months). Normal LDH and absolute lymphocyte count ≥1,000 at 7 weeks predicted longer OS. Immune-related adverse events (irAEs) were noted in 16 patients including 7 with grade 3/4 irAEs (20%)., Conclusion: Ipilimumab is clinically active in acral melanoma with similar ORR and OS compared with unselected melanoma populations. Ipilimumab remains a viable therapeutic option for patients with advanced acral melanoma., Implications for Practice: Ipilimumab is a commonly used immune therapy that improves survival in metastatic melanoma. The clinical activity of ipilimumab in certain rare melanoma subtypes, such as uveal or mucosal melanomas, is suboptimal. Acral melanoma is another unusual subtype of this disease that arises on the palms, soles, and nailbeds. In this study of 35 patients with acral melanoma from 2 centers, ipilimumab was found to have activity that appears equivalent to unselected melanoma (response rate of 11.4%, median overall survival of 16.7 months). Ipilimumab remains a viable treatment option for this melanoma subpopulation., (©AlphaMed Press.)

Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content. [ABSTRACT FROM AUTHOR]

Background: Racial and geographic factors seem to affect the incidence of cutaneous and mucosal melanoma. Objective: To investigate the occurrence of BRAF and cKIT impairments in patients with sinonasal melanoma in Southern Italy. Methods: Eleven sinonasal melanomas were screened for BRAF mutations and cKIT alterations by immunohistochemistry (CD117), fluorescence in situ hybridization and sequencing analyses. Results: A high prevalence (4/11; 36%) of BRAF mutations and lack of cKIT mutations were observed. Amplification of cKIT was found in 18% of cases; cKIT expression was detectable in 18% non-overlapping cases. No correlation between CD117 and cKIT alterations was observed. One (6%) cKIT and two (12%) BRAF mutations were detected in an additional series of 17 acral/mucosal melanomas from the same geographic areas. Conclusion: Mutations of cKIT are infrequent in sinonasal melanoma in Southern Italy. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cutaneous melanoma is a highly aggressive cancer with still limited, but increasingly efficacious, standard treatment options. Recent preclinical and clinical findings support the notion that cutaneous melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma already has great implications for the management of cutaneous melanoma. Herein, we review our rapidly growing understanding of the molecular biology of cutaneous melanoma, including the pathogenic roles of the mitogen-associated protein kinase (MAPK) pathway, the phosphatidylinositol 3 kinase [PI3K]/phosphatase and tensin homologue deleted on chromosome 10 [PTEN]/Akt/mammalian target of rapamycin [mTOR])PTEN (phosphatase and tensin homolog) pathway, MET (hepatocyte growth factor), Notch signaling, and other key molecules regulating cell cycle progression and apoptosis. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with clinical benefit from agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Cutaneous melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. These findings suggest that prospective genotyping of patients with melanoma, combined with the growing availability of targeted agents, which can be used to rationally exploit these findings, should be used increasingly as we work to develop new and more effective treatments for this devastating disease. [ABSTRACT FROM AUTHOR]

Abstract: Loss of cKit receptor in cutaneous melanomas was attributed to the down-regulation of AP2 transcription factor. Our analysis of 27 melanoma cell lines showed no correlation between AP2 and c-kit expression. Suggesting a post-transcriptional mechanism of cKit down-modulation, we performed genome-wide microRNA (miRNA) expression profiling and found that several miRNA species are commonly up-regulated in melanomas. Among them was mir-221, which can directly interact with c-kit 3′UTR and inhibit cKit protein translation. Observed inverse correlation of the c-kit and mir-221 expression in various melanocytic cells pointed to its involvement in regulation of cKit in melanoma. Moreover, a series of functional assays demonstrated that mir-221 could directly inhibit cKit, p27Kip1 and, possibly, other pivotal proteins in melanoma. Collectively, the studies presented here indicate that mir-221 could be a novel therapeutic target for the treatment of cutaneous melanoma. They also suggest that regulation of expression and functional activity of identified up-regulated miRNAs should be further studied in the context of malignant melanoma. [Copyright &y& Elsevier]