Next-Generation Sequencing for UM
Comprehensive genomic profile of an individual UM tumor from a single biopsy
The DecisionDx-UMSeq test is a 7-gene panel that uses next-generation sequencing (NGS) to identify somatic mutations relevant to uveal melanoma (UM). The test includes hotspot mutations in the genes GNAQ, GNA11, CYSLTR2, PLCB4, and SF3B1, mutations in exons 1-2 of EIF1AX, and all coding exons of the BAP1 gene.
This information, along with results from the DecisionDx-UM and DecisionDx-PRAME gene expression profile (GEP) tests, can help to build a comprehensive genomic profile of an individual UM tumor from a single biopsy. The genomic information can be used now to inform patient care and may become useful in the future as research and therapeutics evolve.
DecisionDx-UMSeq can be ordered for patients who are having DecisionDx-UM GEP testing. Castle Biosciences has optimized the sequencing so that it can be run using the same fine-needle aspiration biopsy (FNAB) tissue specimen submitted for GEP testing.
Mutations in four genes affect G-protein-coupled receptor signaling
In UM, mutations in GNAQ, GNA11, CYSLTR2, or PLCB4 result in constitutive activation of G-protein-coupled receptor signaling pathways, such as MAPK, PI3K, PKC, Hippo, etc. (Figure 1) (Van Raamsdonk, 2009; 2011; Johansson, 2016; Moore, 2016).
While some of these pathways may be targeted with inhibitory therapies, these mutations are not currently markers for drug response. These mutations are also not known to be prognostic.
The majority of UM tumors will have a mutation in GNAQ or GNA11, while CYSLTR2 and PLCB4 mutations are less common. These mutations are usually mutually exclusive. The presence of one of these mutations may provide some confidence that a melanocytic tumor was sampled. However, other eye lesions can have these mutations, and some UM tumors will not have a mutation in any of these four genes. These mutations should not be used exclusively to rule-in or rule-out a UM diagnosis in the absence of other clinical/pathological features.
Mutations in three genes are associated with differential clinical outcomes
Mutations occurring in EIF1AX, SF3B1, and BAP1 are usually mutually exclusive:
- The EIF1AX protein is involved in translation (from mRNA to protein) initiation
- SF3B1 is a component of the spliceosome, which regulates transcript usage
- BAP1 is a tumor suppressor gene on chromosome 3 that encodes for a deubiquitinating enzyme that forms protein complexes with BRCA1, BARD1, and ASXL1
It is well known that mutations in BAP1 in UM are associated with a higher risk of metastasis and there is considerable overlap with a Class 2 gene expression profile (GEP) (Harbour, 2010). Some BAP1 mutations are germline and can be inherited (Abdel-Rahman, 2011; Gupta, 2015), but the majority are somatic and thus confined to the tumor.
In retrospective studies, having an EIF1AX or SF3B1 mutation has been shown to be associated with a better prognosis than having a BAP1 mutation (Martin, 2013; Ewens, 2014; Decatur, 2016; Furney, 2013; Harbour, 2013; Yavuzyigitoglu, 2016). Tumors with EIF1AX mutations may have the lowest risk compared to those with SF3B1 mutations, who may experience late metastasis.
Importantly, in a recent retrospective study comparing the prognostic value of these three mutations with the DecisionDx-UM GEP test, a Class 2 result was the only significant, independent predictor of metastasis and UM-related mortality in multivariate analysis (Decatur, 2016). Therefore, the GEP test currently provides the best prognostic information about the tumor.
DecisionDx-UMSeq is available for patients undergoing DecisionDx-UM GEP testing or those who have already received their GEP results. DecisionDx-UMSeq can be run from the same biopsy taken for the DecisionDx-UM GEP test.
A separate DecisionDx-UMSeq requisition form must be completed.
Castle Biosciences will not bill your patient’s insurance for the DecisionDx-UMSeq test; direct payment via a credit card authorization form is required at the time of test order. Contact Castle Biosciences Customer Services at 866-788-9007, option 1 for more information.
Understanding the DecisionDx-UMSeq Results
The DecisionDx-UMSeq test results can be expected in 4 to 6 weeks after receipt of the sample in our laboratory.
The DecisionDx-UMSeq report will tell you if clinically relevant mutations (variants) were found in any of the 7 gene targets. For each mutation found, the report will describe:
- Genomic location of the mutation (where in the gene did it occur)
- Type of mutation (missense, nonsense, etc.)
- Functional change that occurs because of the mutation (i.e. an amino acid change in the protein)
- Frequency that the mutation was detected in the sample (variant allele frequency)
- Potential consequences of that mutation on gene function and relevant literature references
The Tier and Level of Evidence will also be described for each mutation, as recommended by the College of American Pathologists (CAP), the American Society of Clinical Oncology (ASCO), and Association for Molecular Pathologists (AMP) [Li 2017].
BAP1 germline testing information
If your patient has a detected BAP1 mutation and is interested in germline testing, Castle Biosciences can provide information to assist with finding testing services and genetic counseling. Please call Castle Biosciences at 866-788-9007, option 1 for these resources.
Abdel-Rahman MH, Pilarski R, Cebulla CM, et al. Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers. J Med Genet 2011;48:856-9
Aoude LG, Vajdic CM, Kricker A, et al. Prevalence of germline BAP1 mutation in a population-based sample of uveal melanoma cases. Pigment Cell Melanoma Res 2013;26:278-9
Decatur CL, Ong E, Garg N, et al. Driver mutations in uveal melanoma: associations with gene expression profile and patient outcomes. JAMA Ophthalmol 2016;134:728-33
Ewens KG, Kanetsky PA, Richards-Yutz J, et al. Chromosome 3 status combined with BAP1 and EIF1AX mutation profiles are associated with metastasis in uveal melanoma. Invest Ophthalmol Vis Sci 2014;55:5160-7
Furney SJ, Pedersen M, Gentien D, et al. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov 2013;3:1122-9
Gupta MP, Lane AM, DeAngelis MM, et al. Clinical characteristics of uveal melanoma in patients with germline BAP1 mutations. JAMA Ophthalmol 2015;133:881-7
Harbour JW, Onken MD, Roberson ED, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330:1410-3
Harbour JW, Roberson ED, Anbunathan H, et al. Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat Genet 2013;45:133-5
Johansson P, Aoude LG, Wadt K, et al. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4. Oncotarget 2016;7:4624-31
Martin M, Masshofer L, Temming P, et al. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat Genet 2013;45:933-6
Moore AR, Ceraudo E, Sher JJ, et al. Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma. Nat Genet 2016;48:675-80
Njauw CN, Kim I, Piris A, et al. Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneous-ocular melanoma families. PLoS One 2012;7: e35295
Onken MD, Worley LA, Long MD, et al. Oncogenic mutations in GNAQ occur early in uveal melanoma. Invest Ophthalmol Vis Sci 2008;49:5230-4
Rai K, Pilarski R, Cebulla CM, et al. Comprehensive review of BAP1 tumor predisposition syndrome with report of two new cases. Clin Genet 2016;89:285-94
Shoushtari AN and Carvajal RD. GNAQ and GNA11 mutations in uveal melanoma. Melanoma Res 2014;24:525-34
Van Raamsdonk CD, Bezrookove V, Green G, et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009;457:599-602
Van Raamsdonk CD, Griewank KG, Crosby MB, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010;363:2191-9
Yavuzyigitoglu S, Koopmans AE, Verdijk RM, et al. Uveal melanomas with SF3B1 Mutations: a distinct subclass associated with late-onset metastases. Ophthalmology 2016;123:1118-28