Characterization of the Melanoma miRNAome by Deep Sequencing

Background

MicroRNAs (miRNAs) are 18–23 nucleotide non-coding RNAs that regulate gene expression in a sequence specific manner. Little is known about the repertoire and function of miRNAs in melanoma or the melanocytic lineage. We therefore undertook a comprehensive analysis of the miRNAome in a diverse range of pigment cells including: melanoblasts, melanocytes, congenital nevocytes, acral, mucosal, cutaneous and uveal melanoma cells.

Methodology/Principal Findings

We sequenced 12 small RNA libraries using Illumina''s Genome Analyzer II platform. This massively parallel sequencing approach of a diverse set of melanoma and pigment cell libraries revealed a total of 539 known mature and mature-star sequences, along with the prediction of 279 novel miRNA candidates, of which 109 were common to 2 or more libraries and 3 were present in all libraries.

Conclusions/Significance

Some of the novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers to assist in the early detection of distant metastases by measuring the circulating levels in blood. Follow up studies of the functional roles of these pigment cell miRNAs and the identification of the targets should shed further light on the development and progression of melanoma.     

Cytomorphology of Metastatic Melanoma—Use of S-100 Protein In the Diagnosis of Amelanotic Melanoma

The cytomorphological features of cells from 52 cases of metastatic melanoma obtained by fine needle aspiration cytodiagnosis were studied. Morphologically, 11, 19 and 22 cases were classified as spindle, epithelial, and mixed cell types of metastatic melanoma respectively. There were 34 melanotic and 18 amelanotic melanomas. Besides melanin, the presence of intranuclear cytoplasmic inclusions, eosinophilic macronucleoli and giant cells were helpful in the diagnosis of a melanoma. Where attempted, staining for S-100 protein was positive in all the 19 cases (eight amelanotic and 11 sparsely pigmented melanomas). In addition eight cases of metastatic tumour where a differential diagnosis of poorly differentiated carcinoma or large cell lymphoma was entertained, were also studied for localization of S-100 protein and all were found to be negative. Electron microscopy was performed in five cases and showed the presence of melanosomes and/or premelanosomes.     

Cost-Effectiveness of Treatment Strategies for BRAF-Mutated Metastatic Melanoma

Purpose

Genetically-targeted therapies are both promising and costly advances in the field of oncology. Several treatments for metastatic melanoma with a mutation in the BRAF gene have been approved. They extend life but are more expensive than the previous standard of care (dacarbazine). Vemurafenib, the first drug in this class, costs $13,000 per month ($207,000 for a patient with median survival). Patients failing vemurafenib are often given ipilimumab, an immunomodulator, at $150,000 per course. Assessment of cost-effectiveness is a valuable tool to help navigate the transition toward targeted cancer therapy.

Methods

We performed a cost-utility analysis to compare three strategies for patients with BRAF+ metastatic melanoma using a deterministic expected-value decision tree model to calculate the present value of lifetime costs and quality-adjusted life years (QALYs) for each strategy. We performed sensitivity analyses on all variables.

Results

In the base case, the incremental cost-effectiveness ratio (ICER) for vemurafenib compared with dacarbazine was $353,993 per QALY gained (0.42 QALYs added, $156,831 added). The ICER for vemurafenib followed by ipilimumab compared with vemurafenib alone was $158,139. In sensitivity analysis, treatment cost had the largest influence on results: the ICER for vemurafenib versus dacarbazine dropped to $100,000 per QALY gained with a treatment cost of $3600 per month.

Conclusion

The cost per QALY gained for treatment of BRAF+ metastatic melanoma with vemurafenib alone or in combination exceeds widely-cited thresholds for cost-effectiveness. These strategies may become cost-effective with lower drug prices or confirmation of a durable response without continued treatment.     

A Systemic Complete Response of Metastatic Melanoma to Local Radiation and Immunotherapy

BACKGROUND: Melanoma is a relatively immunogenic tumor, in which infiltration of melanoma cells by T lymphocytes is associated with a better clinical prognosis. We hypothesized that radiation-induced cell death may provide additional stimulation of an anti-tumor immune response in the setting of anti-CTLA-4 treatment. METHODS: In a pilot melanoma patient, we prospectively tested this hypothesis. We treated the patient with two cycles of ipilimumab, followed by stereotactic ablative radiotherapy to two of seven hepatic metastases, and two additional cycles of ipilimumab. RESULTS: Subsequent positron emission tomography-computed tomography scan indicated that all metastases, including unirradiated liver lesions and an unirradiated axillary lesion, had completely resolved, consistent with a complete response by RECIST. CONCLUSION: The use of radiotherapy in combination with targeted immunotherapy as a noninvasive in vivo tumor vaccine strategy appears to be a promising method of enhancing the induction of systemic immune responses and anti-tumor effect.     

Fine Needle Aspiration Cytology and Immunocytochemistry of Metastatic Melanoma

The cytological and immunological findings of 81 metastatic melanomas are described. Fine needle aspiration was performed from secondary deposits in lymph nodes (38), subcutaneous and soft tissue (36), abdomen (5), lung (1) from 67 patients with histologically verified malignant melanoma. One patient had disease which had spread into the cerebrospinal fluid. Cytomorphologically the cases were classified as classical (47%), carcinoma-like (22%), spindle cell type (14%), lymphoma like (6%), undifferentiated (6%), myxoid type (3%), and clear cell type (2%). All cases were immunologically characterized using antibodies to S-100, vimentin and cytokeratin. All cases were S-100 positive and the majority (96%) reacted with antibodies to vimentin. A weak heterogenous reactivity to cytokeratin antibody was detected in only eight cases. The HMB45 antibody was applied to 20 cases and 16 (80%) of these tumours were positive. In summary, we found that an immunological characterization was necessary to conclusively diagnose over 50% of metastatic melanomas which presented with an equivocal cytological picture.     

Analysis of BRAF Mutation in Primary and Metastatic Melanoma

Mutation of BRAF has been proposed to contribute to melanoma development. However, it remains unclear whether or not BRAF mutation is associated with any particular stage of melanoma progression. Tumor biopsy specimens from patients with melanoma were analyzed to determine whether the frequency of BRAF mutation in metastatic melanoma differed from primary melanoma. BRAF mutation was present in 15 of 23 (61%) patients with primary melanoma and in 7 of 12 (58%) patients with metastatic melanoma. These results suggest that BRAF mutation in melanoma is most likely to occur prior to the development of metastatic disease.     

Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.The American Cancer Society estimates that in 2009 there will be 68,720 new cases of melanoma in this country with ∼8,650 deaths. Recent studies have demonstrated that the non-canonical Wnt pathway, also known as the Wnt/Ca²⁺ pathway, plays an important role in increasing the metastatic potential of melanoma cells (1–5). Studies from our laboratory demonstrated that increasing Wnt5A, which mediates the non-canonical Wnt/Ca²⁺ signaling pathway, increased melanoma metastasis (1–3), and silencing Wnt5A levels via siRNA³ decreased invasion (2, 3). In addition, we have shown that Wnt5A acts via protein kinase C (PKC) to mediate the motility of melanoma cells via the inhibition of metastasis suppressors and an initiation of the epithelial to mesenchymal transition, characterized by the loss of E-cadherin and the up-regulation of Snail (2).Wnt signaling can be mediated by heparan sulfate proteoglycans (HSPGs) which are important signal transduction modulators. They mediate fibroblast growth factor, Hedgehog, epidermal growth factor, transforming growth factor-β, and WNT signaling pathways (6–11). HSPGs consist of two types, cell surface and basement membrane-associated HSPGs (12). Cell surface HSPGs are glycoproteins with covalently attached unbranched and modified sugar chains known as glycosaminoglycans (GAGs). There are two types of cell surface HSPGs, known as glypicans and syndecans (11, 13). Glypicans are attached to the cell surface via a glycosylphosphatidylinositol anchor, whereas syndecans are type 1 transmembrane proteins. HSPG GAG side chains are unbranched chains of modified repeating disaccharide units of N-acetylglucosamine and glucuronic acid. They are joined to the core protein via a tetrasaccharide linker attached to a serine residue. Following synthesis, these chains undergo modification with the addition of sulfates by N- and O-sulfation (14). The sulfation status determines to which specific portion of the GAG chains ligands, such as Wnt, will attach. The heparan sulfate endosulfatases Sulf1 and Sulf2 are cell surface enzymes that control growth factor signaling. The regulation of the 6-O-sulfation states by these endosulfatases changes the affinity of the GAG chains for ligand binding (15–17). Following sulfation modification, HSPGs can regulate signaling by dimerization (with other HSPGs or canonical signaling receptors), stabilization, or transport of the ligand to or away from the high affinity receptors (18–20). In addition, studies have suggested that the core proteins themselves may also play an important role in cell phenotype and function (21).HSPGs have been implicated in a number of pathological conditions, such as Simpson-Golabi-Behmel overgrowth syndrome (22), fibrodysplasia ossificans progressiva (23), and Alzheimer disease (24). In addition, HSPGs are overexpressed in many forms of cancer, including prostate cancer and melanoma (25, 26). Importantly, in cancer, proteoglycans can have both tumor-promoting and tumor-suppressing activities. This depends on the type of protein core, the GAGs attached, and the localization of the proteoglycan and the molecules they associate with. In addition, the tumor subtype, stages, and degree of tumor differentiation also affect the function of HSPGs (27). HSPGs are cleaved by heparanases or heparin lyases (heparinases), which have been shown to have differing effects on tumor cell activity. For example, treating cancer cells with heparanase-1, which cleaves heparin-like regions (specifically HLGAG sites with O-sulfated l-iduronic acid residues), results in an increase in both tumor growth and metastatic dissemination (28). However, treating tumor cells with heparinase III, which more specifically cleaves HSPGs (i.e. unsulfated d-glucorinic acid, heparan-sulfate-like regions) results in an inhibition of their metastatic capacity (29). Importantly, heparanase I cleaves only certain side chains, where heparinase III treatment cleaves the entire backbone of the HSPG. It is likely that cleavage of specific side chains facilitates cell motility by releasing cells from adhesion to neighboring cells, whereas cleavage of the entire molecule decreases the availability of secreted ligands to their receptors, especially those involved in autocrine signaling, such as Wnt5A.Historically, Wnt5A has been quite difficult to purify from cell culture media, despite the fact that it is a secreted protein. Further, in melanoma cells, Wnt5A appears to be signaling in an autocrine fashion (1, 2). These two observations, together with the fact that Wnt5A undergoes glycosylation (30), led us to hypothesize that HSPGs might be involved in increasing the availability of Wnt5A to its receptor, resulting in an increase in autocrine signaling and ultimately an increase in cellular invasion. In this study, we explore this hypothesis and investigate the role of HSPGs in the Wnt5A signaling cascade in metastatic melanoma cells.     

Protein Profiling of Preeclampsia Placental Tissues

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.     

iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors

Background

Uveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis.

Methods

Eight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch’s membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors.

Results

Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens.

Conclusions

The present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.     

Metastatic Urethral Melanoma: Case Report and Review of the Literature

Melanoma is a cancer that originates from melanocytes, is predominant in adults with white skin, represents 4% of skin cancers, and has high possibility of forming metastasis. This review reports on the case of a young man, age 36 years, previously diagnosed with melanoma. The patient complained of obstructive urinary symptoms and, while he was undergoing a cystoscopy, it was discovered that he had a lesion corresponding with metastatic melanoma of the prostatic urethra, which occluded almost the entire urethra and resulted in blocked urinary flow. He underwent a transurethral resection of the prostate, followed by resection of the lesion. After the procedure, he had good urinary flow and is currently on follow-up.Key words: Melanoma, Urethra, Urinary obstruction, Metastasis, Urethral melanomaPrimary malignant melanoma of the urethra is rare, representing < 1% of all melanomas^1,2; it is often misdiagnosed, which leads to delays in treatment.² The lethality is high, but its incidence is low. Prognosis is considered good if it is detected in its early stages.¹ In recent years, there have been great improvements in patient survival rates. In developed countries, the average estimated 5-year survival is 73%, whereas in developing countries, the average survival is 56%. The estimated world average is 69%.¹ Risk factors in order of importance are sensitivity to the sun, light skin, excessive sun exposure, history of skin cancer, family history of melanoma, congenital nevi, maturity, xeroderma pigmentosum, and dysplastic nevi.¹Individual management according to the clinical presentation is based on extrapolation of evidence for other melanoma treatments.² Due to low occurrence rates of urethral melanoma, the optimal therapy has not yet been established, and surgery remains the mainstay of primary therapy; adjuvant locoregional and systemic therapies are needed.² This article reports on the case of a young patient with metastatic melanoma in the urethra which led to urinary obstruction and urinary symptoms.     

PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.     

A Protein Nuclear Extract from D. melanogaster Larval Tissues

Preparation of protein nuclear extracts is often the first step to study in vitro biological processes occurring in the nucleus of the eukaryotic cell. Nuclear extracts have been extensively used in different model organisms to identify and study protein function in nuclei. Drosophila embryos can be collected in large quantities and have been the source of choice for the production of protein nuclear extracts. However, most of Drosophila in vivo studies on protein function are conducted in larval tissues. Here we report a new method to produce highly stable large-scale protein nuclear extracts from whole Drosophila larvae that are suited for a variety of biochemical analyses.