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. Gαq 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the European Union's Horizon 2020 research and innovation program under grant agreement number 667787 (UM Cure 2020, R.J. Nell and N.V. Menger). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Leiden University Medical Center Biobank Committee and Medisch Ethische Toetsingscommissie under no. B14.003/DH/sh and B20.026. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.