Cluster analysis of multiplex ligation-dependent probe amplification data in choroidal melanoma.

PurposeTo determine underlying correlations in multiplex ligation-dependent probe amplification (MLPA) data and their significance regarding survival following treatment of choroidal melanoma (CM).MethodsMLPA data were available for 31 loci across four chromosomes (1p, 3, 6, and 8) in tumor material obtained from 602 patients with CM treated at the Liverpool Ocular Oncology Center (LOOC) between 1993 and 2012. Data representing chromosomes 3 and 8q were analyzed in depth since their association with CM patient survival is well-known. Unsupervised k-means cluster analysis was performed to detect latent structure in the data set. Principal component analysis (PCA) was also performed to determine the intrinsic dimensionality of the data. Survival analyses of the identified clusters were performed using Kaplan-Meier (KM) and log-rank statistical tests. Correlation with largest basal tumor diameter (LTD) was investigated.ResultsChromosome 3: A two-cluster (bimodal) solution was found in chromosome 3, characterized by centroids at unilaterally normal probe values and unilateral deletion. There was a large, significant difference in the survival characteristics of the two clusters (log-rank, p<0.001; 5-year survival: 80% versus 40%). Both clusters had a broad distribution in LTD, although larger tumors were characteristically in the poorer outcome group (Mann-Whitney, p<0.001). Threshold values of 0.85 for deletion and 1.15 for gain optimized the classification of the clusters. PCA showed that the first principal component (PC1) contained more than 80% of the data set variance and all of the bimodality, with uniform coefficients (0.28±0.03). Chromosome 8q: No clusters were found in chromosome 8q. Using a conventional threshold-based definition of 8q gain, and in conjunction with the chromosome 3 clusters, three prognostic groups were identified: chromosomes 3 and 8q both normal, either chromosome 3 or 8q abnormal, and both chromosomes 3 and 8q abnormal. KM analysis showed 5-year survival figures of approximately 97%, 80%, and 30% for these prognostic groups, respectively (log-rank, p<0.001). All MLPA probes within both chromosomes were significantly correlated with each other (Spearman, p<0.001).ConclusionsWithin chromosome 3, the strong correlation between the MLPA variables and the uniform coefficients from the PCA indicates a lack of evidence for a signature gene that might account for the bimodality we observed. We hypothesize that the two clusters we found correspond to binary underlying states of complete monosomy or disomy 3 and that these states are sampled by the complete ensemble of probes. Consequently, we would expect a similar pattern to emerge in higher-resolution MLPA data sets. LTD may be a significant confounding factor. Considering chromosome 8q, we found that chromosome 3 cluster membership and 8q gain as traditionally defined have an indistinguishable impact on patient outcome

MicroRNAs and Uveal Melanoma: Understanding the Diverse Role of These Small Molecular Regulators.

Uveal melanoma (UM) is a rare tumour of the eye, characterised by a high propensity to metastasise in half of all patients, most frequently to the liver. Although there are effective treatment options for the primary tumour, once metastasis has occurred prognosis is poor, with overall survival limited to months. Currently, there are no effective treatments for metastatic UM, despite the tumour having a well-defined signalling pathway to which many therapies have been directed. In an effort to develop novel treatment approaches, understanding the role of other signalling molecules, such as microRNAs, is fundamental. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in posttranscriptional gene regulation, resulting in reduced target gene expression and subsequent protein translation. In UM, several dysregulated miRNAs have been proposed to play a functional role in disease progression, whereas others have been put forward as clinical biomarkers of high-risk disease following isolation from blood, plasma and exosomes. Most recently, analyses of large datasets have identified promising prognostic miRNA signatures and panels. This review navigates the plethora of aberrant miRNAs disclosed so far in UM, and maps these to signalling pathways, which could be targeted in future therapies for the disseminated disease

Spatial Analysis Made Easy with Linear Regression and Kernels

Kernel methods are a popular technique for extending linear models to handle non-linear spatial problems via a mapping to an implicit, high-dimensional feature space. While kernel methods are computationally cheaper than an explicit feature mapping, they are still subject to cubic cost on the number of points. Given only a few thousand locations, this computational cost rapidly outstrips the currently available computational power. This paper aims to provide an overview of kernel methods from first-principals (with a focus on ridge regression) and progress to a review of random Fourier features (RFF), a method that enables the scaling of kernel methods to big datasets. We show how the RFF method is capable of approximating the full kernel matrix, providing a significant computational speed-up for a negligible cost to accuracy and can be incorporated into many existing spatial methods using only a few lines of code. We give an example of the implementation of RFFs on a simulated spatial data set to illustrate these properties. Lastly, we summarise the main issues with RFFs and highlight some of the advanced techniques aimed at alleviating them. At each stage, the associated R code is provided

CD166(high) Uveal Melanoma Cells Represent a Subpopulation With Enhanced Migratory Capacity

Purpose: Cancer stem cells (CSCs) are a subpopulation of cells with the capacity to drive tumor growth. While there is evidence of the existence of CSCs in uveal melanoma (UM), there is no consensus on their defining markers. In this study, we examined putative CSC markers in UM cell lines, primary UM (PUM), and normal choroidal melanocytes (NCM). Methods: Nonadherent sphere assays were used to assess the tumorigenic potential of 15 PUMs, 8 high (M3) and 7 low (D3) metastatic risk. Flow cytometry was used to compare the expression of CSC markers between 10 PUMs and 4 NCMs, as well as in 8 UM cell lines grown under adherent and nonadherent conditions. Based on the data generated and from TCGA analyses, CD166 was investigated in detail, including its effect on cell migration using a tumor transendothelial migration assay. Results: M3 PUM had a greater melanosphere-forming efficiency than D3 PUM. CD166 and Nestin expression was upregulated in PUM compared to NCM by flow cytometry. UM cell lines resistant to anoikis had increased levels of CD271, Nestin, and CD166 compared with adherent cells. TCGA analysis showed that patients with higher CD166 expression had a poorer prognosis: this was supported by a Mel270 CD166high subpopulation that had enhanced migratory capabilities compared with CD166low cells. IHC showed that CD166 is expressed in the cytoplasm and cell membrane of PUM cells. Conclusions: UM contain a population of cells with characteristics of CSCs. In particular, CD166high UM cells appear to represent a subpopulation with enhanced migratory capacity