Germline copy number variants (CNVs) and somatic copy number alterations (SCNAs) are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit.

Spitzoid neoplasms are a group of melanocytic tumours with distinctive histopathological features. They include benign tumours (Spitz naevi), malignant tumours (spitzoid melanomas) and tumours with borderline histopathological features and uncertain clinical outcome (atypical Spitz tumours). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbour kinase fusions of ROS1 (17%), NTRK1 (16%), ALK (10%), BRAF (5%) and RET (3%) in a mutually exclusive pattern. The chimeric proteins are constitutively active, stimulate oncogenic signalling pathways, are tumourigenic and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz naevi, 56% of atypical Spitz tumours and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signalling of the fusion proteins in vitro. In summary, kinase fusions account for the majority of oncogenic aberrations in spitzoid neoplasms and may serve as therapeutic targets for metastatic spitzoid melanomas.

In melanoma, as well as in other solid tumors, the cells within a given tumor exhibit strong morphological, functional and molecular heterogeneity that might reflect the existence of different cancer cell populations, among which are melanoma-initiating cells (MICs) with 'stemness' properties and their differentiated, fast-growing progeny. The existence of a slow-growing population might explain the resistance of melanoma to classical chemotherapies that target fast growing cells. Therefore, elucidating the biologic properties of MICs and, more importantly, the molecular mechanisms that drive the transition between MICs and their proliferating progeny needs to be addressed to develop an efficient melanoma therapy. Using B16 mouse melanoma cells and syngeneic mice, we show that the inhibition of microphthalmiaassociated transcription factor (Mitf), the master regulator of melanocyte differentiation, increases the tumorigenic potential of melanoma cells and upregulates the stem cell markers Oct4 and Nanog. Notably, p27, the CDK inhibitor, is increased in Mitf-depleted cells and is required for exacerbation of the tumorigenic properties of melanoma cells. Further, a slow-growing population with low-Mitf level and high tumorigenic potential exists spontaneously in melanoma. Ablation of this population dramatically decreases tumor formation. Importantly, these data were confirmed using human melanoma cell lines and freshly isolated human melanoma cell from lymph node and skin melanoma metastasis. Taken together our data, identified Mitf and p27 as the key molecular switches that control the transition between MICs and their differentiated progeny. Eradication of low-Mitf cells might be an appealing strategy to cure melanoma.