Molecular profiling of driver events in metastatic uveal melanoma

Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments. Here we genomically profile metastatic tumors and infiltrating lymphocytes. BAP1 alterations are overrepresented and found in 29/32 of cases. Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease. One outlier tumor has a high mutational burden associated with UV-damage. CDKN2A deletions also occur, which are rarely present in primaries. A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains. Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant. This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease.


Statistics
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n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
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Data analysis
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:  Fig. 1-4, Supplementary Fig. 1-3 and Supplementary Fig. 5 Tumors from 32 patients were studied. The majority of the patients were previously enrolled in a clinical trial, although the present study does not intend to describe the outcome of that trial, but rather to genomically profile the tumors. The tumors were included based on availability of material. Although a greater number of tumors would provide added power for new discoveries, interesting findings could potentially also arise from a single individual. Therefore, prior sample size calculations were not applicable in this case.
No patients were excluded from the study. Some analyses required paired genomic and transcriptomic data, the latter of which was not available for two samples, leading to their exlusion from those analyses. In a single-cell analysis comprising a large number of cells, some were excluded due to quality issues, as defined by default settings in the tool (CellRanger) used for pre-processing.
Three replicates were used to test for differences in cells with reinserted functional BAP1. RNA-seq alignments, Western blot, immunohistochemsitry and proteomics confirmed sucessful viral reintroduction in all three. Genes of interest differing between the conditions with and without this gene reintroduced were successfully validated on subsequent RT-qPCR-experiments, also using three replicates. 6-nearest neighbor transcriptomic classification of a specific tumor was carried out against a 9583 tumors from TCGA, and all six top ranking correlating tumors agreed on a single classification. The method was complemented by mutation analysis of DNA and RNA from the same sample, together sucessfully ruling out a sample mixup. In a study of genes of relevance in recurrent broad copy number changes, genes of interest were compared between datasets from ours and two other studies, validating many of the ranking as top candidates. In assays to test cell proliferation and viability effects upon siRNA knockdown of candidate genes, three biological replicates were used.
No predefined experimental groups were used in this study.
Animal experiments were performed by a technician that was blinded to the experimental design. qRT-PCR analyses were not blinded since they were screens to identify potentially important genes. The genes identified did not have any bearing on the overall interpretation of the data so blinding was not needed. The BAP1 qRT-PCR analysis was performed by two different operators yielding the same results, one of which was blinded to the results from the RNA-seq data.
The following antibodies were used for surface staining: CD3

Animals and other organisms
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Wild animals
Field-collected samples

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.
All flow cytometry antibody clones were well-established clones sold by two or more companies. Antibodies were titrated inhouse and validated by flow cytometry at the indicated dilution factors. For IHC, antibodies used were validated at the Clinical pathology lab of Sahlgrenska Hospital by staining tissue or tumor slides known to be positive or negative for the proteins. The stainings presented were performed in parallell with clinical routine stainings using an Autostainer. The Western blot antibodies were validated based on known sizes of the BAP1 and beta-actin proteins (Supplemental Figure 5e).
The cell line was generated from a tumor that grew up in a PDX model (UM22). Its validation by exome sequencing and RNA sequencing is described in the manuscript.
As described in the manuscript, the cell line and original tumor were both sequenced.
Mycoplasma control was performed regularly using a PCR method. Only mycoplasma negative cell lines were used in the study.

None
The mice were housed in the pathogen-free animal facility of University of Gothenburg. Mice were kept in cages with individual ventilation at ambient temperature (21-23 degrees Celsius) and 20-40% humidity. Mice were given free access to food and water. The dark-ligh cycle was 12 hrs dark and light, respectively (dark 7pm to 7am). 6-8 week old female NOD/SCID/Il2R-gamma knockout mice (NOG/NSG) mice were used in the experiments.
No wild animals were used.
No field-collected samples were used.
Regional animal ethics committee of Gothenburg approval #36-2014. 32 metastatic tumors from patients diagnosed with uveal melanoma, six subcutaneous and 26 from the liver, were collected from 14 males and 18 females with an average age of 65 years and a range of 34-80. Twenty-eight of the corresponding primary tumors were pathologically designated as originating from the choroid, one from the ciliary body, one from the iris, and two unknown. All liver biopsies came from patients that were untreated at the time of biopsy and eighteen of them had been enrolled in the SCANDIUM phase III clinical trial of IHP. All cutaneous metastases came from patients that had been treated with chemotherapy (IHP, dacarbazine and/or taxanes).