TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours

Background:

Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations.

Methods:

The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.

Results:

Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma.

Conclusion:

Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.     

Melanoma-expressed CD70 is involved in invasion and metastasis

Background:

CD70 is a costimulatory molecule of the tumour necrosis factor family expressed in activated immune cells and some solid tumours. In lymphocytes CD70 triggers T cell-mediated cytotoxicity and mitogen-activated protein kinase phosphorylation.

Methods:

We evaluated the expression of CD70 in biopsies and melanoma cell lines. Using melanoma cell lines positive or not for CD70, we analysed CD70 function on melanoma progression.

Results:

We report CD70 expression in human melanoma cell lines and tumour cells from melanoma biopsies. This expression was observed in 95% of primary melanomas but only 37% of metastases. Both monomeric and trimeric forms of CD70 were detected in tumour cell membrane fractions, whereas cytoplasmic fractions contained almost exclusively monomeric CD70. In vitro and in vivo experiments demonstrated that CD70 expression inhibited melanoma cell migration, invasion and pulmonary metastasis implantation independently of the tumour immune microenvironment. Increasing the levels of the trimeric form of CD70 through monoclonal antibody binding led to an increase in CD70+ melanoma cell invasiveness through MAPK pathway activation, RhoE overexpression, ROCK1 and MYPT1 phosphorylation decrease, and stress fibres and focal adhesions disappearance.

Conclusions:

Our results describe a new non-immunological function of melanoma-expressed CD70, which involves melanoma invasiveness through MAPK pathway, RhoE and cytoskeletal modulation.     

Differential expression of microRNAs during melanoma progression: miR-200c, miR-205 and miR-211 are downregulated in melanoma and act as tumour suppressors

Background:

The incidence of malignant melanoma is increasing faster than that for any other cancer. Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis.

Methods:

To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs). Differential expression was validated by qRT–PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression.

Results:

In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas. In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion.

Conclusion:

We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.     

Role of EIF5A2, a downstream target of Akt,in promoting melanoma cell invasion

Background:

Cutaneous melanoma is a life-threatening skin cancer because of its poorly understood invasive nature and high metastatic potential. This study examines the importance of eukaryotic translation initiation factor 5A2 (EIF5A2) in melanoma pathogenesis.

Methods:

We examined EIF5A2 expression in 459 melanocytic lesions using tissue microarray. In addition, melanoma cell lines were subjected to invasion and cell proliferation assays, zymography, FACS and real-time PCR to investigate the role of EIF5A2 in cancer progression.

Results:

Positive EIF5A2 staining increased from dysplastic naevi to primary melanomas (PMs; P=0.001), and further increased in metastatic melanomas (P=0.044). Eukaryotic translation initiation factor 5A2 expression was correlated with melanoma thickness (P<0.001) and was inversely correlated with the 5-year survival of PM patients especially those with tumour⩽2 mm thick. Strikingly, none of the latter died within 5 years in EIF5A2-negative staining group. Cox regression analysis revealed that EIF5A2 is an independent prognostic marker. Further, we found that EIF5A2 is a novel downstream target of phosphorylated Akt. Both melanoma cell invasion and MMP-2 activity increased and decreased with EIF5A2 overexpression and knockdown, respectively.

Conclusion:

We for the first time showed that EIF5A2, as a target of PI3K/Akt, promotes melanoma cell invasion and may serve as a promising prognostic marker and a potential therapeutic target for melanoma.     

Expression and functional role of CRIPTO-1 in cutaneous melanoma

Background:

CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma.

Methods:

Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers.

Results:

Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth.

Conclusion:

These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.     

Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion

Background:

Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined.

Results and methods:

Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal–epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT–PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival.

Conclusion:

TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.     

Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma

Background:

Current therapies for metastatic melanoma are targeted either at cancer mutations driving growth (e.g., vemurafenib) or immune-based therapies (e.g., ipilimumab). Tumour progression also requires angiogenesis, which is regulated by VEGF-A, itself alternatively spliced to form two families of isoforms, pro- and anti-angiogenic. Metastatic melanoma is associated with a splicing switch to pro-angiogenic VEGF-A, previously shown to be regulated by SRSF1 phosphorylation by SRPK1. Here, we show a novel approach to preventing angiogenesis—targeting splicing factor kinases that are highly expressed in melanomas.

Methods:

We used RT–PCR, western blotting and immunohistochemistry to investigate SRPK1, SRSF1 and VEGF expression in tumour cells, and in vivo xenograft assays to investigate SRPK1 knockdown and inhibition in vivo.

Results:

In both uveal and cutaneous melanoma cell lines, SRPK1 was highly expressed, and inhibition of SRPK1 by knockdown or with pharmacological inhibitors reduced pro-angiogenic VEGF expression maintaining the production of anti-angiogenic VEGF isoforms. Both pharmacological SRPK1 inhibitors and SRPK1 knockdown reduced growth of human melanomas in vivo, but neither affected cell proliferation in vitro.

Conclusions:

These results suggest that selective blocking of pro-angiogenic isoforms by inhibiting splice-site selection with SRPK1 inhibitors reduces melanoma growth. SRPK1 inhibitors may be used as therapeutic agents.     

Host-derived MMP-13 exhibits a protective role in lung metastasis of melanoma cells by local endostatin production

Background:

Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood.

Methods:

Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT–PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied.

Results:

Lung metastases of B16BL6 cells were significantly higher by 2.5–5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin.

Conclusion:

Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.     

Societal preference values for advanced melanoma health states in the United Kingdom and Australia

Background:

No studies measure preference-based utilities in advanced melanoma that capture both intended clinical response and unintended toxicities associated with treatment.

Methods:

Using standard gamble, utilities were elicited from 140 respondents in the United Kingdom and Australia for 13 health states.

Results:

Preferences decreased with reduced treatment responsiveness and with increasing toxicity.

Conclusions:

These general population utilities can be incorporated into treatment-specific cost-effectiveness evaluations.     

Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma

Background:

Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin.

Methods:

GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH.

Results:

Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS.

Conclusion:

The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.     

The melanoma-specific graded prognostic assessment does not adequately discriminate prognosis in a modern population with brain metastases from malignant melanoma

Background:

The melanoma-specific graded prognostic assessment (msGPA) assigns patients with brain metastases from malignant melanoma to 1 of 4 prognostic groups. It was largely derived using clinical data from patients treated in the era that preceded the development of newer therapies such as BRAF, MEK and immune checkpoint inhibitors. Therefore, its current relevance to patients diagnosed with brain metastases from malignant melanoma is unclear. This study is an external validation of the msGPA in two temporally distinct British populations.

Methods:

Performance of the msGPA was assessed in Cohort I (1997–2008, n=231) and Cohort II (2008–2013, n=162) using Kaplan–Meier methods and Harrell''s c-index of concordance. Cox regression was used to explore additional factors that may have prognostic relevance.

Results:

The msGPA does not perform well as a prognostic score outside of the derivation cohort, with suboptimal statistical calibration and discrimination, particularly in those patients with an intermediate prognosis. Extra-cerebral metastases, leptomeningeal disease, age and potential use of novel targeted agents after brain metastases are diagnosed, should be incorporated into future prognostic models.

Conclusions:

An improved prognostic score is required to underpin high-quality randomised controlled trials in an area with a wide disparity in clinical care.     

Q-TWiST analysis comparing ipilimumab/dacarbazine vs placebo/dacarbazine for patients with stage III/IV melanoma

Background:

Study CA184024 was a multinational, randomised, double-blind, phase 3 study comparing ipilimumab/dacarbazine (DTIC) vs placebo/DTIC in patients with untreated stage III/IV melanoma, which showed that ipilimumab significantly improves survival in patients with metastatic melanoma. The objective of this analysis was to compare the quality-adjusted survival experience among patients in this trial.

Methods:

Survival time was partitioned into health states: toxicity, time before progression without toxicity, and relapse until death or end of follow-up. Q-TWiST (quality-adjusted time without symptoms of disease or toxicity of treatment) was calculated as the utility-weighted sum of the mean health state durations. Analyses were repeated over extended follow-up periods.

Results:

Based on a combination of trial-based and external utility scores, the Q-TWiST difference in this trial was 0.50 months (P=0.0326) favoring ipilimumab after 1 year. The Q-TWiST difference was 1.5 months with 2 years of follow-up (P=0.0091), 2.36 months at 3 years (P=0.005) and 3.28 months at 4 years (P=0.0074).

Conclusion:

During the first year of study, there was little difference between groups in quality-adjusted survival. However, after 2, 3 and 4 years follow-up for patients with extended survival, the benefits of IPI+DTIC vs PLA+DTIC for advanced melanoma continue to accrue.     

ING4 regulates JWA in angiogenesis and their prognostic value in melanoma patients

Background:

We previously showed that inhibitor of growth family member 4 (ING4) inhibits melanoma angiogenesis, and JWA suppresses the metastasis of melanoma cells. As angiogenesis is essential for tumour metastasis, further investigation of the function of ING4 and JWA in melanoma angiogenesis is needed, and their prognostic value are of great interest.

Methods:

Western blot, tube-formation assays and luciferase assays were used to investigate the correlation between ING4 and JWA in melanoma angiogenesis. JWA and integrin-linked kinase (ILK) expression was determined on a tissue microarray constructed from 175 biopsies.

Results:

ING4 promoted JWA expression by activating JWA promoter. Furthermore, the regulation of growth and tube formation of endothelial cells by ING4 was partially JWA dependent. Also, ING4 inhibited the ILK-induced angiogenesis signalling pathway via JWA. Moreover, reduced JWA, or increased ILK, expression was closely associated with 5-year disease-specific survival of melanoma patients (P=0.001 and 0.007, respectively). There was also a positive correlation between ING4 and JWA yet a negative correlation between ING4 and ILK. Importantly, their concomitant expressions were significantly related to 5-year survival of melanoma patients (P=0.002 and 0.003, respectively).

Conclusion:

JWA has an important role in ING4-regulated melanoma angiogenesis, and ING4/JWA/ILK are promising prognostic markers and may be used as anti-angiogenic therapeutic targets for melanoma.     

miR-21 and miR-155 are associated with mitotic activity and lesion depth of borderline melanocytic lesions

Background:

Expression of microRNAs (miRs) has been shown to be altered in many solid tumours and is being explored in melanoma. The malignant potential of some melanocytic lesions is difficult to predict. We hypothesised that characterisation of miR expression in borderline melanocytic proliferations would lead to the identification of a molecular profile that could be used with known prognostic factors to differentiate lesions with high malignant potential.

Methods:

The miR expression profile of melanocytic lesions (benign naevi, malignant melanoma and borderline melanocytic tumours) was evaluated by real-time PCR.

Results:

PCR analysis revealed primary cutaneous melanomas had an 8.6-fold overexpression of miR-21 and a 7.5-fold overexpression of miR-155 compared with benign naevi (P<0.0001). In situ hybridisation confirmed these results. miR-21 and miR-155 were significantly overexpressed within borderline lesions (P=0.0011 and P=0.0048, respectively). When borderline lesions were categorised by mitotic activity and Breslow thickness, miR-21 was associated with mitotic activity and miR-155 was associated with thickness (P<0.025). Among 14 patients with borderline lesions who underwent sentinel lymph node biopsy (SLNB), positive SLNB was associated with increased miR-21 and miR-155 in the primary lesion compared with lesions with a negative SLNB.

Conclusion:

MicroRNA expression profiles can be used to characterise atypical melanocytic lesions.     

KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab

Background:

We aimed to compare the sensitive and quality-controlled KRAS testing with direct sequencing and to assess the impact on decision making of treatment.

Patients and methods:

We analysed genomic DNA isolated from macrodissected formalin-fixed paraffin-embedded specimens by direct sequencing and an amplification refractory mutation system–Scorpion assay (ARMS/S) method. Cetuximab was administered to patients identified as having wild-type (WT) KRAS using direct sequencing. Therapeutic effects were evaluated according to their KRAS status as determined by ARMS/S.

Results:

Among the 159 patients, the overall mutation rate was determined to be 37.0% by direct sequencing and 44.0% by ARMS/S. For the patients diagnosed as WT by direct sequencing and treated with cetuximab (n=47), a response rate of 16.0% was observed for 38 ARMS/S WT patients, whereas 9 ARMS/S mutant (MUT) patients failed to respond. The ARMS/S WT patients showed significant improvement in progression-free survival (PFS) and overall survival (OS) compared with ARMS/S MUT patients (PFS median 5.0 vs 1.7 months, hazards ratio (HR)=0.29, P=0.001; OS median 12.1 vs 3.8 months, HR=0.26, P=0.001).

Conclusion:

Sensitive and quality-controlled KRAS testing may provide improved predictive power to determine the efficacy of anti-epidermal growth factor antibodies.