Digital PCR-based deep quantitative profiling delineates heterogeneity and evolution of uveal melanoma

Uveal melanoma is an aggressive intraocular tumour characterised by a limited number of genetic alterations. However, the evolution of this malignancy remains enigmatic. In this study, we performed a deep quantitative analysis of 80 primary uveal melanomas by novel digital PCR-based approaches. Mutations were quantified by targeted and drop-off mutation assays, copy number alterations were precisely measured by quantifying the allelic imbalance of heterozygous single-nucleotide polymorphisms. By comparing the absolute abundances of genetic alterations present in a bulk tumour, the heterogeneity and early evolution could be inferred. Tumour progression was further studied by analysing matched primary and metastatic lesions from five patients. Gq signalling mutations were generically and always clonally present, suggesting to be acquired in the earliest stage of uveal melanoma development ( primary driver). Next, three main evolutionary subtypes could be identified based on having an EIF1AX mutation, SF3B1 mutation or monosomy 3p. These alterations were usually mutually-exclusive and (near-) clonally abundant, suggesting to represent distinct secondary drivers. This contrasts with gains and amplifications of chromosome 8q, which were not restricted to one of the main subtypes and showed subclonality in 31% of the affected tumours. These tertiary alterations were not required for metastatic dissemination. Using high-resolution analyses, we identified systematic differences in the evolutionary timing of genetic events in uveal melanoma. The observed intratumour heterogeneity suggests a more complex model of gradual tumour evolution and argues for a comprehensive genetic analysis in clinical practice, which may be facilitated by the sensitive digital PCR assays developed in this study.