DOI: 10.5061/dryad.z8w9ghxqk
title: Data from: Early genetic evolution of driver mutations in uveal melanoma

description: Uveal melanoma (UM) is an aggressive cancer of the eye that frequently
 results in metastatic death. UMs are most likely to metastasize when they
 are small, at a time when they are difficult to distinguish from benign
 nevi and often observed without treatment. Unfortunately, little is known
 about the early genetic evolution of UM or potential biomarkers to
 indicate small tumors undergoing malignant transformation. Here, we
 performed targeted next generation sequencing for the 7 canonical UM
 driver mutations in 1140 primary UMs, including 131 small early-stage
 tumors. We found that the evolutionary burst of genetic aberrations that
 determines the archetypal UM subtypes and metastatic propensity has
 already occurred by the time most small tumors are biopsied, although a
 significantly larger proportion of small tumors are still evolving
 compared to larger tumors. We found that the 15-gene expression profile
 (15-GEP) support vector machine discriminant score was the best indicator
 of tumors in transition from low-risk Class 1 to high-risk Class 2
 signature. While BAP1, SF3B1, and EIF1AX mutations were associated with
 poor, intermediate, and good prognosis, respectively, mutation analysis
 was inferior to the prospectively validated 15-GEP + PRAME expression
 classifier for predicting metastasis-free and overall survival. These
 results provide a more complete picture of genetic evolution in UM, and
 they move us closer to a molecular definition of malignant transformation
 in this cancer type.
description: # Data from: Early genetic evolution of driver mutations in uveal melanoma
 Dataset DOI: [10.5061/dryad.z8w9ghxqk](10.5061/dryad.z8w9ghxqk) ##
 Description of the data and file structure ### Files and variables ####
 File: DryadUpload_SupplementaryInformationSData1-4_v14.xlsx
 **Description:** The "Description" tab outlines contents, along
 with important abbreviations and details for the Supplementary Data. The
 tabs following the Description tab include Supplementary Data 1-4 (large
 data tables from the manuscript). Supplementary Data 1 includes a table of
 baseline clinical annotations, patient outcomes, and genetic features for
 all study subjects (n = 1140 cases). Supplementary Data 2 includes
 statistical analysis of uveal melanoma-associated mutations for all study
 subjects (n = 1140 cases). Supplementary Data 3 includes statistical
 analysis of small tumors (n = 131 cases) versus larger tumors (n = 1009
 cases). Supplementary Data 4 includes statistical analysis of patients
 with BAP1-mutant tumors (n = 364) versus those with BAP1-wildtype tumors
 (n = 776). ##### Variables * Includes Supplementary Data 1, 3, 6, and 7.
 #### File: DryadUpload_SourceData_v4.xlsx **Description:** Source Data
 file contains tables of raw data used to generate all plots in main and
 Supplementary Figures. The "Description" tab describes each
 dataset and includes additional column descriptions that may be helpful
 for understanding included data. Exact p-values included for all
 significance testing displayed in figures. Order of tables by figure
 (panels): Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 2a, Figure
 2b, Figure 2c, Figure 2d, Figure 3a-f, Figure 4a, Figure 4b, Figure 4c,
 Figure 4d, Figure 4e, Supplementary Figure 2, Supplementary Figure 3,
 Supplementary Figure 4, Supplementary Figure 5, Supplementary Figure 6,
 Supplementary Figure 7 ##### Variables * Excel file with each spreadsheet
 containing source data for data presented in manuscript. #### File:
 forcedCallVCFs_chr3p_coog2.2_998pts.zip **Description:** The
 dataset consists of a directory of forced called VCF files from targeted
 hybrid capture of each patient within the cohort for copy number calls of
 BAP1 (n=998). ##### Variables * ZIP folder of VCF files. ## Patient
 Enrollment This research complies with all relevant ethical regulations.
 Federal Wide Assurance (FWA) from the Office of Human Research Protections
 (OHRP) and Institutional Review Board (IRB) or Ethics Committee approval
 was obtained in accordance with policies at each participating center,
 with oversight by the University of Miami IRB. Between January 2017 and
 April 2020, COOG2 enrolled 1687 subjects with UM involving the choroid,
 ciliary body and/or iris across 26 ocular oncology centers in the U.S. and
 Canada and prospectively monitored these subjects for metastatic
 progression and outcome. Informed written consent was obtained from each
 patient. Primary treatment was performed according to the standard at each
 center. Exclusion criteria included patient age less than 18 years,
 diagnosis of a uveal tumor other than UM (e.g., metastatic cancer), prior
 radiotherapy, inadequate sample for molecular analysis, and patient
 withdrawal from the study. Prior photodynamic therapy or transpupillary
 thermotherapy was allowed if there was evidence of tumor regrowth. No
 participants were excluded based on sex, ethnicity, or race. Gender was
 recorded from medical records and used as a proxy for biological sex in
 this study. No additional data on gender identity was collected. For this
 analysis, a data lock was performed on March 4, 2024. Subjects were not
 included for this report if they had a primary iris melanoma (n = 101
 cases), lacked adequate residual biopsy material for successful sequencing
 (n = 212 cases) or had no detectable UMAM (UM-associated mutations) (n =
 234 cases). ## Description of Data Data includes prognostic testing
 results of primary UM from DecisionDx®-UM 15-gene expression profile
 (15-GEP), which renders a result of Class 1 or Class 2, and
 DecisionDx®-PRAME, which renders a result of negative or positive. The
 15-GEP test employs support vector machine (SVM) to classify each sample
 as Class 1 or Class 2 and to assign a discriminant score as a measure of
 confidence in the Class call based on the distance of a given sample to
 the decision boundary. The residual material was analyzed with the custom
 UMAM NGS (next-generation sequencing) panel DecisionDx®-UMSeq. Genetic
 variants were included that were classified as tier I, II, or III
 according to the guidelines of the College of American Pathologists (CAP),
 American Society of Clinical Oncology (ASCO), and Association for
 Molecular Pathology (AMP). This testing was performed in a CAP-accredited,
 CLIA-certified clinical laboratory (Castle Biosciences, Inc., Friendswood,
 TX, USA). Mutations were classified as nonsense (introduction of a
 premature stop codon), stop-loss or start-loss (loss of stop or start
 codon preventing translation), frameshift insertion or deletion (shift of
 codon reading frame via addition or subtraction of a non-triplet set of
 nucleotides), non-frameshift insertion or deletion (addition or removal of
 a codon without shifting the reading frame), block substitution
 (alteration of multiple sequential codons), splice site alteration
 (alteration of splice donor or acceptor site), missense (substitution of
 one amino acid).The following BAP1 variants were called pathogenic:
 nonsense, stop-loss, start-loss, frameshift and non-frameshift insertions
 and deletions, block substitutions, and splice site alterations. The
 remaining (predominantly missense) variants were called pathogenic if
 they: (1) were previously reported as pathogenic in the ClinVar Database
 32, (2) exhibited a SIFT score less than or equal to 0.05, or (3)
 exhibited a PolyPhen2 score greater than or equal to 0.5. Tumor purity
 (TP), the percentage of cells in a sample that are tumor cells, was
 inferred from the variant allele frequency (VAF) of the Gq mutation,
 assuming that the Gq mutation is the founder mutation, is a heterozygous
 alteration, and is therefore present at 50% VAF in tumor cells. In rare
 cases with more than one Gq mutation, the mutation with the highest
 frequency (and presumably the earlier initiating mutation) was used. As
 such, TP=min([VAF-Gq-mutant x 2],100%). The variant allele fraction (VAF)
 for BAP1, SF3B1, and EIF1AX mutations was corrected for TP using the
 following equation: TP-corrected VAF-BSE = VAF-BSE ÷ TP. Samples without a
 detectable Gq mutation could not be corrected for VAF and, thus, were not
 included in analyses requiring TP-corrected VAF-BSE. Next, we estimated
 the cancer cell fraction (CCF) for each BSE mutation, representing the
 proportion of UM cells that harbor a given mutation, which requires a
 correction for allelic copy number. SF3B1 is located on chromosome 2,
 which is not frequently altered in UM20,22. Thus, SF3B1 mutations were
 assumed to be heterozygous and CCF-SF3B1 = min (TP-corrected VAF-SF3B1 x
 2, 100%). EIF1AX is located on the X chromosome, which is also rarely lost
 in UM. Thus, gender was used to calculate mutant CCF-EIF1AX, where females
 were assumed to have an EIF1AX mutation at 50% and males at 100% of
 TP-corrected VAF. Thus, the CCF-EIF1AX for females was calculated as
 CCF-EIF1AX=min(TP-corrected VAF-EIF1AX x 2, 100%), whereas the CCF-EIF1AX
 for males was assumed to be equal to TP-corrected VAF-EIF1AX. BAP1 is
 located at chromosome 3p2133, which frequently undergoes copy number loss
 in UM. To detect loss of heterozygosity (LOH) and calculate CCF for BAP1,
 we developed a custom targeted CNV sequencing panel containing 74 loci
 across chromosome 3p that was performed on the same sample used for the
 15-GEP/PRAME classifier and UMAM NGS panel (description below). For
 BAP1-mutant tumors with retention of heterozygosity for chromosome 3p, the
 CCF-BAP1 was calculated as CCF-BAP1=min(TP-corrected VAF-BAP1 x 2, 100%).
 For tumors demonstrating LOH for chromosome 3p (LOH3p), CCF-BAP1 was
 assumed to be equal to TP-corrected VAF-BAP1. For the custom CNV
 sequencing panel, B-allele frequencies and log fold-change (lfc) read
 depths across chromosome 3p were compared to a reference DNA panel of
 normals (PON), comprising peripheral blood mononuclear cell (PBMC) samples
 from 64 patients. Variant call format (VCF) files were analyzed using
 Wheeljack
 ([https://github.com/covingto/KRCGTK/releases/tag/v0.1](https://github.com/covingto/KRCGTK/releases/tag/v0.1)). Copy-number loss for chromosome 3p was detected by consistent b-allele frequencies at 100% and a decreased lfc read depth of less than 0. Isodisomy for chromosome 3p was identified by consistent b-allele frequencies at 100% and a lfc read depth of approximately 0. For downstream analyses, samples demonstrating either copy number loss or isodisomy for chromosome 3p were called as LOH3p, whereas samples without these aberrations were called as retention of heterozygosity for 3p. Calls were made by hand and adjudicated by 3 authors. Variability across b-allele and read depth plots was used to assign confidence scores with 0, 1, 2, and 3 corresponding to very low, low, medium, and high confidence, respectively. A confidence score of 2 or 3 was required for use in downstream analyses.