Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through wholegenome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy.

Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.

The diagnosis of salivary gland neoplasms is a complex and difficult area of surgical pathology. Most of the salivary gland tumours present a varieted and pleomorphic morphology that make difficult the histopathological categorisation. In addition, there are no clinical and/or radiological features specific for each neoplasm. According to the latest WHO classification of the salivary gland tumours, there are more than 30 benign and malignant neoplastic entities, 1 15%-26% are malignant [2][3][4] and approximately 85% originate from the parotid gland. 5 Fine-needle aspiration (FNA) has been used for more than 50 years for the diagnosis of salivary gland tumours. 6 FNA is a safe, quick, less invasive, cost effective and relatively reliable diagnostic tool, especially when performed under the ultrasound guidance 7 and in experienced and skilful hands, 8 can be easily performed in the outpatient setting. In addition, it helps to plan the management and extent of parotid surgery. 8 However, FNA presents a high false positive rate, 9,10 and the data of its sensitivity, specificity and accuracy are controversial. FNA sensitivity presents a huge variability between 41.7% and 92.8%, and its specificity is 93.9%-98.5%. The overall diagnostic accuracy rates vary from 79% to 97%. 11,12 For malignant neoplasms, the FNA diagnosis is nondiagnostic in 26% of the cases. 11,13 In a series of 151 cases, FNA results were non-diagnostic/inconclusive in 18% of cases 14 and its sensitivity was 76%-92% for benign tumours and 53%-79% for malignant neoplasm. [15][16][17][18] FNA specimens are inadequate for diagnosis in 9%-12% of all cases and in 14% of malignant tumours. 18,19 It is worth mentioning that FNA is operator dependent technique and the results are strongly related to the experience of the clinicians and cytopathologists.

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Metastatic melanoma carries a poor prognosis despite modern systemic therapies.Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal that metastatic cells may depart the primary tumour very early in the disease course and follow a branched pattern of evolution. Truncal UV-induced mutations that often swamp downstream analyses of heterogeneity, were found to be replaced by APOBEC-associated mutations in the branches of the evolutionary tree. Multi-sample analyses from a further 7 patients confirmed that branched evolution was pervasive, representing an important mode of melanoma dissemination. Our analyses illustrate that combining cancer cell fraction estimates across multiple metastases provides higher resolution phylogenetic reconstructions relative to single sample analyses and highlights the limitations of accurately inferring inter-tumoural heterogeneity from a single biopsy.